D-enantiomer peptides, compositions including the same and uses thereof for treatment of liver disorders

Abstract

Provided herein are isolated D-peptides having one or more D-amino acids, capable of affecting the neuroligin-4 (NLGN4)-neurexin 1β (Nrx1β) protein-protein interaction, wherein the D-peptide has improved human serum stability, enhanced ability to reduce activation of Hepatic Stellate Cells (HSCs) and / or attenuate their proliferation. Further provided are compositions including such peptides and uses thereof for treating and / or attenuating liver-related conditions.

Description

[0001] D-ENANTIOMER PEPTIDES, COMPOSITIONS INCLUDING THE SAME AND USES THEREOF FOR TREATMENT OF LIVER DISORDERS FIELD OF THE INVENTION

[0002] The present invention relates to novel D-enantiomeric peptides capable of reducing activity of human hepatic stellate cells (HSCs) and / or affecting neuroligin-4 (NLGN4)-neurexin ip (Nrxip) protein-protein interactions, compositions including the same and uses thereof in treating and / or attenuating liver-related conditions and / or various types of cancer.

[0003] BACKGROUND OF THE INVENTION

[0004] The liver is a complex organ, composed of hepatocytes and non-parenchymal cells, which includes Kupffer cells, endothelial cells, myofibroblasts known as Hepatic Stellate Cells (HSCs) and leukocytes. In healthy livers, HSCs are in a quiescent state, but when the liver is chronically injured, HSCs are transformed into an activated state characterized by enhanced proliferation and production of excessive extracellular matrix (ECM), as part of the pathogenesis of liver fibrosis. Natural killer (NK) cells play a critical role in the early stages of the immune innate response. NK cells participate in the process of liver fibrosis and serve as anti-fibrotic activity through HSCs killing, but their function decreases when the liver disease progresses into cirrhosis in a long-standing disease.

[0005] During these advanced stages of liver fibrosis liver NK cells exert an overexpression of adhesion molecules called NLGN4, this overexpression correlates to the less killing activity of NK cells, while HSCs in the same liver are overexpressing NLGNN4 adhesion partner: -NRXN. This reflects an interplay that contributes to the progression of liver fibrosis and is considered to involve the NLGN / Nrxi axis between HSCs and NK cells that leads to NK cells inhibition pathway.

[0006] The disruption of this interplay has been the focus of two earlier publications. International application, publication No. WO 2016 / 157195 relates to inhibitors of NLGN / Nrxip protein-protein interaction for treatment of liver disorders.

[0007] International application, publication No. WO 2023 / 170678 relates to peptide modulators of neuroligin 4-neurexin 1-beta axis for treatment of liver disorders.However, while interfering with the NLGN4 / NrxlBeta axis effectively enhances the activity of NK cells and its killing of HSCs in vitro and in vivo, peptide-based therapeutics (of less than 50 amino acids in length and less than 10 kDa in molecular weight), particularly small, isolated, L-amino acid based therapeutic peptides are prone to low metabolic stability or poor pharmacokinetics.

[0008] The lower the molecular weight of the peptide, the shorter the life span. Thus, a peptide with a molecular weight of less than 4 kDa is susceptible to proteolytic degradation and would circulate in plasma just a few minutes before it is degraded.

[0009] A peptide drug with a half-life of only few minutes is not expected to be clinically effective, because it would not deliver sufficient drug to the target tissues.

[0010] While structural modification can be applied to increase the half-life of the NLGN4 peptide, even a single modification of just a single amino acid (AA) is expected to result with an altered structure of the peptide that will negatively affect its activity and impair its function.

[0011] There is, therefore, an unmet need for therapeutic peptides with improved metabolic stability or prolonged half-life for use in treating liver related conditions, by affecting the NLGN4-Nrxip interaction in various cells and tissues.

[0012] SUMMARY OF THE INVENTION

[0013] According to some aspects, there are provided herein isolated D-peptide(s), compositions, kits and cells including the same, as well as methods of treatment using the same, wherein the isolated D-peptide(s) includes at least one D amino acid (dAA).

[0014] According to some embodiments, advantageously, the disclosed isolated D-peptide(s) have at least one dAA (also referred to herein as D-peptides), and has improved stability. Improved stability may be demonstrated, for example, in human serum in comparison to a reference peptide having a similar amino acid sequence, but does not include any dAA (also referred to herein as L-peptide).

[0015] In some embodiments, the isolated D-peptide(s) is herein exemplified to have a half-life of at least about 2, 3, 4, 5, 6, 7 days (e.g., 14 days) in human serum, and could be detected at concentrations of about 100 nanomolar (nM) even 24 hours after intravenous injection to mice induced with acute liver fibrosis.Surprisingly, according to some embodiments, the disclosed D-peptides were herein demonstrated to be capable of deactivating human hepatic stellate cells (HSCs), inhibiting proliferation of HSCs, or affecting neuroligin-4 (NLGN4)-neurexin ip (Nrxi ) protein-protein interactions in comparison to untreated cells and / or cells treated with a reference L-peptide (e.g., a peptide not having any D-amino acid). Each possibility is a separate embodiment.

[0016] Thus, in some embodiments, the present disclosure provides compositions and methods to prevent or treat liver disorders and / or cancer, including liver fibrosis and / or liver cancer, utilizing D-peptides characterized by improved stability in human serum and enhanced capability for deactivation of HSCs and attenuation of their proliferation.

[0017] In some embodiments, the isolated D-peptides disclosed herein have an AA sequence as denoted by SEQ ID NO: 1, wherein at least one of the AAs of SEQ ID NO: 1 is a dAA.

[0018] According to one aspect, there is provided an isolated D-peptide comprising an amino acid (AA) sequence as denoted by SEQ ID NO: 1, wherein SEQ ID NO: 1 comprises at least one D enantiomeric AA (dAA).

[0019] According to some embodiments, the D-peptide consisting of an AA sequence as denoted by SEQ ID NO: 1, wherein SEQ ID NO: 1 comprises at least one dAA.

[0020] In some embodiments, SEQ ID NO: 1 may include 2 or more (e.g. 2 to 9) dAA. In some embodiments, all 9 chiral amino acids (AAs) of SEQ ID NO: 1 are D-amino acid (all-dAAs), as denoted by SEQ ID NO: 2.

[0021] In some embodiments, the isolated D-peptide is capable of reducing the activation of Hepatic Stellate Cells (HSCs).

[0022] In some embodiments, the isolated D-peptide is capable of attenuating HSCs proliferation and / or viability. Each possibility is a separate embodiment.

[0023] In some embodiments, the isolated D-peptide has an improved stability in human serum in comparison to a reference peptide that has an amino acid sequence as denoted by SEQ ID NO: 3; wherein all chiral amino acids (AAs) of the reference peptide are L-amino acids (all-lAAs).

[0024] In some embodiments, the isolated D-peptide is stable for at least about 60 minutes (e.g., at least about 120 minutes) at 37°C in serum.In some embodiments, the isolated D-peptide has a half-life of at least 1, 2, 3, 4, 5, 6, 7 days (e.g., at least about 14 days) in serum.

[0025] In some embodiments, the isolated D-peptide has a half-life of at least about 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 90 minutes in vivo.

[0026] In some embodiments, the isolated D-peptide is capable of affecting neuroligin 4 (NLGN4) -Neurexin ip (Nrxi ) protein-protein interaction.

[0027] According to another aspect, there is provided a composition including an isolated D-peptide, and at least one pharmaceutically acceptable carrier / s, excipient / s, auxiliaries, and / or diluent / s. Each possibility is a separate embodiment.

[0028] According to some embodiments, the isolated D-peptide, or the composition including the same, can be used for treating, attenuating, and / or preventing progression of a liver disorder in a subject in need thereof. Each possibility is a separate embodiment.

[0029] In some embodiments, the liver disorder may be selected from: fibrosis, Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD), Metabolic Dysfunction-Associated Steatohepatitis (MASH)cirrhosis, hepatitis, viral hepatitis, liver adenoma, insulin hypersensitivity, liver cancer, liver metastasis, or any combination thereof. Each possibility is a separate embodiment.

[0030] In some embodiments, the liver disorder is liver fibrosis or liver cancer. Each possibility is a separate embodiment.

[0031] According to some embodiments, the isolated D-peptide, or the composition including the same, can be used for treating, attenuating, and / or preventing progression of a cancer condition in a subject in need thereof. Each possibility is a separate embodiment.

[0032] In some embodiments, the cancer may be selected from liver cancer, liver metastasis, cholangiocarcinoma, colorectal cancer, breast cancer and prostate cancer, or any combination thereof. Each possibility is a separate embodiment.

[0033] In some embodiments, the isolated D-peptide or the composition including the same may be used by systemic administration.In some embodiments, the isolated D-peptide or the composition including the same may be administered in combination with at least one additional therapeutic agent.

[0034] In some embodiments, the isolated D-peptide or the composition may be used for reducing activation of HSCs. In some embodiments, the isolated D-peptide or the composition including the same may be used for inhibiting HSCs proliferation. Each possibility is a separate embodiment.

[0035] According to yet another aspect, there is provided a natural killer (NK) cell including the isolated D-peptide, or the composition comprising the same.

[0036] In some embodiments, the NK cell is capable of reducing or inhibiting activity of HSCs. Each possibility is a separate embodiment.

[0037] According to yet another aspect, there is provided a composition including the NK cells.

[0038] In some embodiments, the NK cell or the composition comprising the same, may be used for treating, attenuating and / or preventing progression of a liver disorder in a subject in need thereof. Each possibility is a separate embodiment.

[0039] In some embodiments, the liver disorder may include: fibrosis, Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD), Metabolic Dysfunction-Associated Steatohepatitis (MASH), cirrhosis, hepatitis, viral hepatitis, liver adenoma, insulin hypersensitivity, liver cancer, liver metastasis, or any combination thereof. Each possibility is a separate embodiment.

[0040] In some embodiments, the liver disorder is liver fibrosis or liver cancer. Each possibility is a separate embodiment.

[0041] In some embodiments, the NK cell or the composition including the same, may be for use in treating, attenuating and / or preventing progression of cancer in a subject in need thereof. Each possibility is a separate embodiment.

[0042] In some embodiments, the cancer may be selected from: liver cancer, liver metastasis, cholangiocarcinoma, colorectal cancer, breast cancer and prostate cancer. Each possibility is a separate embodiment.

[0043] In some embodiments, the NK cell or the composition including the same, may be administered systemically. Each possibility is a separate embodiment. Each possibility is a separate embodiment.In some embodiments, the NK cell or the composition including the same, may be administered in combination with at least one additional therapeutic agent. Each possibility is a separate embodiment.

[0044] According to yet another aspect, there is provided a method of treating, attenuating and / or preventing progression of a liver disorder in a subject in need thereof, the method includes administering a therapeutically effective amount of the isolated D-peptide, or the composition including the same. Each possibility is a separate embodiment.

[0045] According to yet another aspect, there is provided a method of treating, attenuating and / or preventing progression of a liver disorder in a subject in need thereof, the method includes administering a therapeutically effective amount of the NK cell or the composition comprising the same. Each possibility is a separate embodiment.

[0046] According to yet another aspect, there is provided a method of treating, attenuating and / or preventing progression of a cancer condition in a subject in need thereof, the method includes administering a therapeutically effective amount of one or more of: the isolated D-peptide or the composition including the same; the NK cell or the composition including the same. Each possibility is a separate embodiment.

[0047] Further embodiments, features, advantages and the full scope of applicability of the present invention will become apparent from the detailed description and drawings given hereinafter. However, it should be understood that the detailed description, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0048] BRIEF DESCRIPTION OF THE FIGURES FIG. 1 presents a bar graph showing the mean fluorescence intensity (MFI) of a-SMA expression in LX2 cells monoculture treated with the reference peptide of SEQ ID NO: 3 (L-peptide), the peptide of SEQ ID NO: 2 (D-peptide), Rczdiffra™ (MGL), a combination of MGL and the reference L-peptide, or with a combination of MGL and the reference D-peptide, and compared to untreated cells (Not treated). After reaching a confluence state LX2 cells were treated with the reference L-peptide, the D-peptide, or MGL by adding each to the culture at final concentrations of 8 pm, 8 pm or 6 nM,respectively, for 24h of incubation before harvesting the monoculture, fixing and permeabilizing the cells, and adding anti a-SMA antibody for further evaluation of a-SMA levels using FACS analyzer, (ns, *, **, *** indicates p>0.05, p=<0.05, p=<0.01, p=<0.001, respectively, as calculated from six technical repetitions).

[0049] FIG. 2 presents a line graph showing the absorbance (490nm) of MTS assay performed on LX2 cells in monoculture treated with the D-peptide of SEQ ID NO: 2 in different final concentrations of 250, 500, 2000, 3000, 4000 and 8000 nM for dose response evaluation of proliferation / viability, and compared to a control treatment of a vehicle solution (Not treated). Following 24-hours of exposure to treatment, the cells were mized with MTS solution and then incubated at 37°C for 2 hours, before absorbance was measured, (p-values are indicated, as calculated from six technical repetitions).

[0050] FIGs. 3A-3C present line graphs showing stability in human serum for the D-peptide (SEQ ID NO: 2) or the reference L-peptide (SEQ ID NO: 3) incubated at 37°C for: 0, 5, 10, 30, 60, or 120 minutes (FIG. 3A); or 0, 0.5, 1, 2, 4, 6, or 24 hours (FIG. 3B), or 0, 2, 3, 6, 12, or 14 days (FIG.3C), before LCMS / MS analysis was performed to assess peptide concentrations at the indicated timepoints.

[0051] FIG. 4A presents line graphs of pharmacokinetic study of D-peptide half life, when administered to healthy mice by IV (Intravenous), IM (Intra muscular), or S.C (Subcutaneous) route of administration;

[0052] FIG. 4B presents a line graph showing the stability and calculated half-life of the isolated D-peptide of SEQ ID NO: 2 in vivo in blood collected from CC14-induced acute hepatic fibrosis model mice intravenously injected with the peptide. LCMS / MS analysis was performed to samples collected at 0, 2, 24 and 48 hours post injection;

[0053] FIG. 5 shows the effect of an all-D peptide (SEQ ID NO: 2), on monocultures of NK cells. The bar graph shows NK degranulation (percentage ofCD107a) in mono-culture of untreated NK cells (NK + NT), or NK-cells treated with the D-peptide (4000 ng / mL).

[0054] FIG. 6 shows bar graphs of quantification of ALT levels (U / L) under the various indicated treatments. Data is shown as mean + SEM. One-way ANOVA followed by Dunnett’s multiple comparisons test;

[0055] FIGs. 7A-7C show bar graphs of the quantification the effect of treatment on various liver mRNA biomarkers. Fig. 7A- MCP-1; Fig. 7B- IL-6; Fig. 7C- TIMP-1. Data isshown as mean + SEM. One-way ANOVA followed by Dunnett’s multiple comparisons test;

[0056] FIGs. 8A-8C show bar graphs of quantification of the effect of treatment on various liver protein biomarkers. Fig.8A- P-Neurexin expression; Fig. 8B- aSMA expression;

[0057] Fig. 8C- LAMP-1. Data is shown as mean + SEM. One-way ANOVA followed by Dunnett’s multiple comparisons test.;

[0058] FIGs. 9A-9D show bar graphs of quantification of the effect of treatment on liver histopathology. Fig. 9A - fibrosis score, Fig. 9B - inflammation; Fig. 9C- Hepatocyte necrosis; Fig. 9D- Hepatocyte vacuolation / degeneration rarefaction. Data is shown as mean + SEM. One-way ANOVA followed by Dunnett’s multiple comparisons test.;

[0059] Fig. 10 shows a histogram of the quantification of flow cytometry analysis of blood samples obtained from various treated mice groups, to determine the presence of CD107aNK-cells.

[0060] DETAILED DESCRIPTION

[0061] In the following description, various aspects of the disclosure will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the disclosure. However, it will also be apparent to one skilled in the art that the disclosure may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

[0062] Definitions:

[0063] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0064] The following are terms that are used throughout the description, and which should be understood in accordance with the various embodiments to mean as follows:As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise, “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the stated object, unless the context clearly dictates otherwise. By way of example, “a D-peptide” means one or more D-peptides(s).

[0065] As used herein, the term "about" or “approx.” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass deviations / variations of ±20% or in some embodiments ±10%, or in some embodiments ±5%, or in some embodiments ±1%, or in some embodiments ±0.1% from the specified value, as such deviations are appropriate to perform the disclosed methods. Each possibility is a separate embodiment.

[0066] As used herein, the term “comprising” is synonymous with the terms "including", "containing", “having”, or "characterized by" and is inclusive or open-ended i.e. does not exclude additional, unrecited elements. According to some embodiments, the term comprising may be replaced with the term “consisting of’ which excludes any element, step, or ingredient not specified in the claim. According to some embodiments, the term comprising may be replaced with the term “consisting essentially of’ which limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristics" of the claimed invention.

[0067] As used herein, the terms “D-peptide” and “D-polypeptide”, “isolated D-peptide”, “D-enantiomer peptide” “all-DAAs peptide”, “interfering D-peptide” are used interchangeably to refer to non-naturally occurring peptides synthetically produced to include one or more D enantiomers of amino acids (dAAs).

[0068] As used herein, the terms “NLGN4-Nrxip protein-protein interaction (PPI) interfering D-peptide”, "NLGN4-Nrxip interfering D-peptides" and “NLGN4-Nrxip protein-protein interaction (PPI) modulator D-peptide” may interchangeably be used. These terms relate to an isolated “D-peptide” (and / or compositions including the same) that can modulate and / or interfere with the NLGN4 and Nrxip interaction. In some embodiments, such peptides have the ability to interact with and / or bind to a site / region in NLGN4 and / or Nrxip, to thereby modulate and / or interfere with the NLGN4 andNrxip interaction. In some embodiments, the interaction and / or modulation / interference is transient. In some embodiments, the interaction and / or modulation / interference is reversible. In some embodiments, the interaction and / or modulation / interference is permanent (irreversible). The interaction / binding of the D-peptide to NLGN4 and / or Nrxip may occur in-vivo, ex vivo or in-vitro. For example, in some embodiments, interaction / binding of the D-peptide to NLGN4 and / or Nrxip may occur in-vivo, in tissue / cells of a subject (for example, NK cells). For example, in some embodiments, interaction / binding of the D-peptide to NLGN4 and / or Nrxip may occur in-vitro, i.e., the D-peptide may bind NLGN4 / Nrxip in NK cells extracted from the subject's body. In some embodiments, such NK cells may later be administered to the patient.

[0069] As used herein, the terms “modulate”, “affect”, “alter” and “interfere”, may be used interchangeably. The phrase “capable of affecting / modulating / interfering” as used herein refers to the capability of an isolated D-peptide to at least partially inhibit or prevent interaction or binding between NLGN4 and Nrxip or to interact with a target of NLGN4 or Nrxip, such that the target becomes less accessible, preferably inaccessible to binding by its corresponding binding partner (for example, Nrxip or NLGN4), for example the receptor's natural antigen. In some embodiments, the isolated D-peptide is capable of at least partially inhibiting binding between NLGN4 and Nrxip.

[0070] In some embodiments, the term “D-peptide” refers to a peptide(s) having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, including, according to some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 D-enantiomeric AAs (dAA). Each possibility is a separate embodiment.

[0071] In some embodiments, the isolated D-peptide has an amino acid sequence as denoted by SEQ ID NO: 2 including 9 chiral amino acids (AAs) synthesized as D-amino acids: [H-( dM )( dE ) ( dQ ) G ( dE ) ( dF ) ( dL ) ( dN ) ( dY )( dD )-OH], therefore, the D-peptide of SEQ ID NO: 2 is also herein referred as all-dAAs D-peptide.

[0072] In some embodiments, a D-peptide refers to a peptide(s) that has a sequence than encompasses the amino acids sequence of SEQ ID NO: 1 or SEQ ID NO: 2, including sequences such as, but not necessarily limited to: QILMEQGEFLNYDIM (as denoted by SEQ ID NO: 4) and / or DPQILMEQGEFLNYDIMLGV (as denoted bySEQ ID NO: 5), wherein SEQ ID NO: 4 or SEQ ID NO: 5 comprise 1, 2, 3, 4, 5, 6, 7, 8, or 9 D-enantiomeric AAs (dAA) selected from the amino acids sequence MEQGEFLNYD that is denoted in SEQ ID NO: 1 or SEQ ID NO: 2. Each possibility is a separate embodiment.

[0073] In some embodiments, the activity of the isolated D-peptide may be compared to a reference peptide - an “isolated L-peptide” - that has a sequence of amino acids that corresponds to the sequence of amino acids of the D-peptide of SEQ ID NOs 1- or 2. The amino acid sequence of the isolated reference L-peptide is denoted by SEQ ID NO: 3 and includes 9 chiral amino acids (AAs) synthesized as L-amino acids (all-lAAs): [MEQGEFLNYD], therefore, the reference L-peptide of SEQ ID NO: 3 is also herein referred as all-lAAs L-peptide.

[0074] As used herein, the terms “reference peptide”, “isolated L-peptide”, “L-peptide” or “all-lAAs peptide” are used interchangeably to refer to a peptide that does not include any dAA. In some embodiments, an L-peptide has an amino acid sequence of SEQ ID NO: 3 which is devoid of any D enantiomeric AA (dAA).

[0075] When there is no indication that D enantiomeric AAs are included, the L isomer / enantiomer was used. The D isomers are indicated by “D” (as in “D-peptide”), or indicated by “d” before the residue abbreviation (as in “dAA”). Alternatively, it is explicitly stated that the AA sequence includes such one or more D isomers / enantiomers of the amino acid (as in “wherein SEQ ID NO: 4 or SEQ ID NO: 5 comprise at least one D enantiomeric AA selected from the amino acids in the sequence MEQGEFLNYD).

[0076] In some embodiments, the D-peptides may be produced by synthetic methods. Synthetic methods include, for example, exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, or classical solution synthesis. Solid phase peptide synthesis procedures are well known to one skilled in the art and described, for example by John Morrow Stewart and Janis Dillaha Young, Solid Phase Polypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984). In some embodiments, synthetic peptides are purified by preparative high-performance liquid chromatography (Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.). The peptide sequence may be confirmed by amino acid sequencing usingmethods known to one skilled in the art. In some embodiments, modified in vitro translation systems may be utilized to produce D-amino acid peptides.

[0077] As used herein, the term “isolated" means: 1) separated from at least some of the components with which it is usually associated in nature; 2) prepared or purified by a process that involves the hand of man; 3) not occurring in nature.

[0078] According to some embodiments, the D-peptides are intended to encompass modified sequences (i.e., the “modified” refers to “variant” and “analog” sequences or peptides, as described herein below).

[0079] In some embodiments, the D-peptides of the invention may include variant sequences. The “variant sequences” of the D-peptide refer to D-peptides with amino acid substitutions, both conservative and non-conservative as described below that have the same or improved activity compared to the reference L-peptide (e.g., denoted by SEQ ID NO: 3) or to any of the D-peptides disclosed herein (e.g., denoted by SEQ ID NO: 1-2 and 4-5).

[0080] According to some embodiments, “Salts” of the peptides contemplated by the invention include physiologically and pharmaceutically acceptable organic and inorganic salts. The invention also provides conservative amino acid variants of the D-peptides. Variants according to the invention may also be made such that the overall molecular structure of the encoded D-peptides is conserved. Given the properties of the individual amino acids comprising the disclosed D-peptide products, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e., “conservative substitutions,” may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved. The amino acids used in this invention are those, which are available commercially or are available by routine synthetic methods. Certain residues may require special methods for incorporation into the D-peptide, and either sequential, divergent or convergent synthetic approaches to the D-peptide sequence are useful in this invention. Natural coded amino acids and their derivatives are represented by three-letter codes according to IUPAC conventions.

[0081] According to some embodiments, conservative substitutions of L- or D-amino acids as known to those skilled in the art are within the scope of the present invention.Conservative L- or D-amino acid substitutions include replacement of one L- or D-amino acid with another having the same type of functional group or side chain, e.g., aliphatic, aromatic, positively charged, negatively charged. These substitutions may enhance oral bioavailability, penetration into the islets, targeting to specific beta cell populations, immunogenicity, and the like. One of skill will recognize that individual substitutions, deletions or additions to a D-peptide, D-polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an L- or D-amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar L- or D-amino acids are well known in the art.

[0082] The following six groups each contain L- or D-amino acids that are conservative substitutions for one another:

[0083] 1- L- or D-Alanine (A), L- or D-Serine (S), L- or D-Threonine (T);

[0084] 2- L- or D-Aspartic acid (D), L- or D-Glutamic acid (E);

[0085] 3- L- or D- Asparagine (N), L- or D-Glutamine (Q);

[0086] 4- L- or D-Arginine (R), L- or D-Lysine (K);

[0087] 5- L- or D- Isoleucine (I), L- or D-Leucine (L), L- or D-Methionine (M), L- or D-Valine (V); and

[0088] 6- L- or D- Phenylalanine (F), L- or D-Tyrosine (Y), L- or D-Tryptophan (W).

[0089] In some embodiments, the D-peptides of the invention may include analog peptides or sequences. The term “analog” refers to a molecule, which has the amino acid sequence according to the invention except for one or more amino acid changes. Analogs according to the present invention may include peptidomimetics. “Peptidomimetic” refers to a peptide modified in such a way that it includes at least one non-coded residue or non-peptidic bond. Such modifications include, e.g., alkylation and more specific methylation of one or more residues, insertion of or replacement of natural amino acid by non-natural amino acids, replacement of an amide bond with another covalent bond. A peptidomimetic according to the present invention may optionally comprise at least one bond, which is an amide -replacement bond such as urea bond, carbamate bond, sulfonamide bond, hydrazine bond, or any other covalent bond. The design of appropriate “analogs” may be computer assisted. Analogs are included in the invention as long as they remain pharmaceutically acceptable.In some embodiments, the D-polypeptide may include one or more amino acid substitutions, additions, or deletions. In some embodiments, the D-polypeptide may include one or more substitutions corresponding to a conservative variant of the L- or D-amino acid.

[0090] In some embodiments, the terms “similarity”, "homology" and “sequence similarity / homology” may be interchangeably used and refer hereinafter to the level of identities between two homologous sequences when they are compared by aligning them using an alignment tool. As used herein, the sequence similarity is with respect to any one of the amino acid sequences disclosed herein and denoted by any one of SEQ ID NO: 1, SEQ ID NO: 2 SEQ ID NO: 4 and / or SEQ ID NO: 5. The level of similarity or homology is determined by the number of identities in the amino acid sequence when they are aligned. In some embodiments, the level of similarity between two amino acid sequences is the degree of identity between residues of the aligned sequence.

[0091] According to some embodiments, the amino acid sequence of the D-peptide of the invention has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 99.9% sequence identity to the amino acid sequence as set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2 SEQ ID NO: 4 and / or SEQ ID NO: 5. Each possibility is a separate embodiment. The term “plurality of isolated D-peptides” refers to a combination / mixtures including one or more of the isolated D-peptides of the invention as set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2 SEQ ID NO: 4 and / or SEQ ID NO: 5 and / or homologs thereof including variant D-peptides having conservative L- or D- amino acid substitutions and / or D-peptide analog thereof.

[0092] Neuroligin-4 (NLG4, NLGN4, NLGn4X) is a family member of neuronal cell surface proteins called the Neuroligins. Members of this family are membrane-anchored proteins acting as ligands for beta-neurexins. Neurexin (NRXN), namely neurexin i (Nrxi ), is a member of the presynaptic protein family (Neuroligins ligands) that help to glue together neurons at the synapse. Neurexins are located mostly on the presynaptic membrane and contain a single transmembrane domain. The extracellular domain interacts with proteins in the synaptic cleft, most notably neuroligin, while the intracellular cytoplasmic portion interacts with proteins associated with exocytosis.In some embodiments, the term “binding site” or “epitope” as used herein refers to the region of NLGN4 and / or Nrxip that specifically reacts with a particular D-peptide.

[0093] In some embodiments, the terms “Neuroligin 4”, “Neuroligin 4X”, “NLGN4” and “NLG4” are interchangeable and as used herein refer to the protein product of the NLGN4 gene e.g., NP_001269075.1, NP_001269074.1, NP_851849.1 and NP 065793.1.

[0094] In some embodiments, the terms “neurexin-l-beta”, “ -neurexin” “Nrxip” and “Nrxnl” are interchangeable and as used herein refer to the protein product of the Nrxip gene e.g., NP_001129131.1 NP_004792.1 NP_620072.1.

[0095] According to some embodiments, the terms “administration” and “administering” refer to providing or giving a subject a therapeutic agent (e.g. an isolated D-peptide or composition comprising the same), by any effective route. In some embodiments, exemplary routes of administration include, but are not limited to, injection or infusion, such as subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intravenous, intracerebroventricular, intrastriatal, intracranial and into the spinal cord, oral, intraductal, sublingual, rectal, topical, transdermal, intranasal, vaginal and inhalation routes. Each possibility is a separate embodiment.

[0096] In some embodiments, the terms “liver disorder”, “liver disease”, “liver-related disorder”, “liver-related condition” and “hepatic disease” are used interchangeably and refer to diseases and disorders that cause the liver to function improperly or to stop functioning. In some embodiments, the liver related condition is a Steatotic Liver Disease (SLD). In some embodiments, the liver related condition may be selected from: non-alcoholic fatty liver disease (NAFLD) (also referred to herein as Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD)), non-alcoholic steatohepatitis (NASH) (also referred to herein as Metabolic Dysfunction-Associated Steatohepatitis (MASH)), Cryptogenic SLD (including patients with liver fat who have no metabolic risk factors and no other known cause for the condition), MetALD (including patients having have MASLD but also consume more alcohol than the strict "non-alcoholic" limit, but not enough to be classified as having Alcoholic Liver Disease (ALD)), cirrhosis, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, liveradenoma, insulin hypersensitivity, liver cancer, liver metastasis, and the like, or any combination thereof. Each possibility is a separate embodiment.

[0097] As used herein, the terms NAFLD (non-alcoholic fatty liver disease) and MASLD (Metabolic Dysfunction-Associated Steatotic Liver Disease) are used interchangeably. The terms generally relate to Hepatic steatosis (liver fat) plus at least one cardiometabolic risk factor (like obesity, Type 2 diabetes, or high blood pressure).

[0098] As used herein, the terms NASH (non-alcoholic steatohepatitis) and MASH (metabolic Dysfunction-Associated Steatohepatitis (MASH) are used interchangeably. The terms encompass an advanced Hepatic steatosis stage involving liver inflammation and damage.

[0099] In some embodiments, the terms “subject” and “patient” are interchangeable and as used herein refer to any individual suffering from, or at risk of developing, a liver disorder, or other related condition, such as cancer.

[0100] In some embodiments, the term “treatment” as used herein refers to both therapeutic treatment and prophylactic or preventative measures. In some embodiments, those in need of treatment include those already having a disorder as well as those in which the disorder is to be prevented. As used herein, the terms “prevent”, “reduce”, “attenuate”, “ameliorate”, “inhibit” may be used interchangeably. As used herein, the terms “enhanced”, “increased”, “improved” “elevated” may be used interchangeably.

[0101] In some embodiments, the terms “composition” and “pharmaceutical composition” are used interchangeably and as used herein refers to any composition including at least one D-peptide of the invention, or a composition which includes cells (such as, NK cell) which include (harbor) the D-peptides. In some embodiments, the composition can include a plurality of D-peptides. In some embodiments, the composition can include a plurality of such cells. In some embodiments, the plurality of D-peptides may be a plurality of the same D-peptide, or a combination of several D-peptides. For example, in some embodiments, the composition may include any one or more of the D-peptides represented by SEQ ID NO: 1.In some embodiments, the term “pharmaceutically acceptable carrier” refers to any carrier conventionally used in the production of pharmaceutical compositions. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition, 1975, describes compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein.

[0102] It should be appreciated that the compositions of the present disclosure, and specifically, the pharmaceutical compositions disclosed herein, may comprise the active compound in free form and be administered directly to the subject to be treated. Alternatively, depending on the size of the active molecule (specifically, the D-peptide), it may be desirable to conjugate it to a pharmaceutically acceptable carrier prior to administration. Therapeutic formulations may be administered in any conventional dosage formulation. Formulations typically comprise at least one active ingredient, as defined above, together with one or more pharmaceutically and physiologically acceptable carriers in the sense of being compatible with the other ingredients and not injurious to the patient .

[0103] In some specific embodiments, the pharmaceutical composition of the present disclosure may be suitable for injection. The pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0104] The pharmaceutical compositions of the present disclosure generally comprise a buffering agent, an agent who adjusts the osmolarity thereof, and optionally, one or more pharmaceutically acceptable carriers, excipients and / or additives as known in the art. Supplementary active ingredients can also be incorporated into the compositions. The carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants .As used herein “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.

[0105] Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable .

[0106] The pharmaceutical compositions of the present disclosure, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0107] According to some aspects, there are provided herein novel isolated D-peptides, compositions including the same and uses thereof for treating liver disorders by modulating, interfering with, inhibiting and / or preventing hepatic stellate cells (HSCs) activation or NLGN4-Nrxip interaction, in particular, by preventing protein-protein interaction ("PPI") thereof.

[0108] In some embodiments, the isolated D-peptides include an amino acid sequence derived from NLGN4X. The D-peptides may include different amino acid sequences that can vary in length, and may be derived, without wishing to be bound by any theory or mechanism of action, from the same binding site as that of P-neurexin (i.e., the ligand of the NLG4NX receptor). Without wishing to be bound by any theory or mechanism of action, in some embodiments, the active binding site may be in a domain containing amino acids 359-364 in NLGN4X protein. In some embodiments, epitopes involved in binding may include, E361, L363, H267, Y463 and E270. In some embodiments, the isolated D-peptides are derived from the E361 site epitope.According to some embodiments the isolated D-peptide is used in a method of treating, attenuating and / or preventing progression of a liver disorder, the method includes administering a therapeutically effective amount of D-peptide (or a composition including the same), the D-peptide has an amino acid sequence as denoted by any one of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5; wherein in some embodiments, said D-peptide is capable of inhibiting or deactivating HSCs or interfering with, inhibiting and / or preventing neuroligin 4 (NLGN4) - Neurexin ip (Nrxi ) interaction. Each possibility is a separate embodiment.

[0109] According to some embodiments, the D-peptide, may be used in combination with at least one additional therapeutic agent, such as, PD1 and PDL1 inhibitors. In some embodiments, the D-peptide may be comprised in a single composition with the additional therapeutic agent.

[0110] According to some embodiments, the D-peptide is a synthetic peptide.

[0111] According to some embodiments, there are provided herein methods and compositions for treating, attenuating, and / or preventing progression of a liver disorder by activating natural killer cells (NK cells) and / or deactivating hepatic stellate cells (HSCs) by utilizing the D-peptide(s) of the invention. Each possibility is a separate embodiment. In some embodiments, the liver disorder may be selected from, but is not limited to: fibrosis, non-alcoholic fatty liver disease (NAFLD or MASLD), nonalcoholic steatohepatitis (NASH or MASH), cirrhosis, hepatitis, viral hepatitis, liver adenoma, insulin hypersensitivity, liver cancer (hepatocellular), liver metastasis, or any combination thereof. Each possibility is a separate embodiment.

[0112] According to some embodiments, there are provided herein methods and compositions for treating, attenuating, and / or preventing progression of cancer condition in a subject in need thereof. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer may be selected from, but is not limited to: liver cancer (hepatocellular), liver metastasis, cholangiocarcinoma, colorectal cancer, breast cancer and prostate cancer, or any combination thereof. Each possibility is a separate embodiment.In some embodiments, the isolated D-peptide includes an amino acid (AA) sequence as denoted by SEQ ID NO: 1, wherein SEQ ID NO: 1 includes at least one D enantiomeric AA (dAA).

[0113] In some embodiments, SEQ ID NO: 1 includes 2 to 9 D- enantiomeric AA (dAA). Each possibility is a separate embodiment.

[0114] According to some embodiments, the isolated D-peptide may have an amino acid sequence as denoted by SEQ ID NO: 1: MEQGEFLNYD wherein SEQ ID NO: 1 includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 D-enantiomeric AA (dAA). Each possibility is a separate embodiment.

[0115] In some embodiments, the isolated D-peptide includes all 9 chiral amino acids (AAs) of SEQ ID NO: 1 as D-amino acid (all-dAAs), as denoted by SEQ ID NO: 2.

[0116] According to some embodiments, the isolated D-peptide may have an amino acid sequence as denoted by SEQ ID NO: 2: (dM)(dE)(dQ)G(dE)(dF)(dL)(dN)(dY)(dD). Each possibility is a separate embodiment.

[0117] According to some embodiments, the isolated D-peptide sequence may have an amino acid sequence as denoted by SEQ ID NO: 4: QILMEQGEFLNYDIM, wherein SEQ ID NO: 4 includes at least one D enantiomeric AA (dAA) selected from the amino acids in the sequence MEQGEFLNYD. In some embodiments, the D-peptide comprises or consists of SEQ ID NO: 4, wherein SEQ ID NO: 4 includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 D enantiomeric AA (dAA) selected from the amino acids in the sequence MEQGEFLNYD. Each possibility is a separate embodiment.

[0118] According to some embodiments, the isolated D-peptide sequence may have an amino acid sequence as denoted by SEQ ID NO: 5: DPQILMEQGEFLNYDIMLGV, wherein SEQ ID NO: 5 includes at least one D enantiomeric AA (dAA) selected from the amino acids in the sequence MEQGEFLNYD. In some embodiments, the D-peptide comprises or consists of SEQ ID NO: 5, wherein SEQ ID NO: 5 includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 D-enantiomeric AA (dAA)D enantiomeric AA (dAA) selected from the amino acids in the sequence MEQGEFLNYD. Each possibility is a separate embodiment.According to some embodiments, the novel D-enantiomer amino acid peptides were generated based on the L-enantiomer amino acid peptides sequences MEQGEFLNYD. Replacing L-enantiomer amino acid with D-enantiomer amino acid may alter the structure of the peptide and disrupt its function. Even replacement of just a single chiral amino acid (AA) in the amino acid sequence with its un-naturally occurring D-enantiomeric AA is expected to result with an altered structure of the peptide that will negatively affect its activity and impair its function.

[0119] Therefore, the activity of the isolated D-peptide was evaluated in cultured HSCs.

[0120] According to some embodiments, as demonstrated herein, it was surprisingly found that, the D-peptides of the invention can reduce activity or deactivate HSCs, as exemplified by profound reduction in aSMA level (alpha Smooth Muscle Actin).

[0121] In some embodiments, the isolated D-peptide is capable of reducing activation of Hepatic Stellate Cells (HSCs). In some embodiments, reduction in HSCs activation comprises reduction in alpha-SMA levels in comparison to untreated cells or cells treated with a reference peptide that has an amino acid sequence as denoted by SEQ ID NO: 3; wherein all chiral amino acids (AAs) of the reference peptide are L-amino acids (all-lAAs). Each possibility is a separate embodiment.

[0122] As used herein, the term “reducing” refers to the ability to decrease by 1-100% an activity, expression, binding, activation, and the like, of genes, proteins, cells, and the like. In some embodiments, reducing encompasses partial or complete, reduction or deactivation. In some embodiments, with respect to reducing activation of Hepatic Stellate Cells (HSCs), the reduction may range from partial activity reduction to complete deactivation.

[0123] In some embodiments, the isolated D-peptide is capable of deactivating Hepatic Stellate Cells (HSCs), said deactivation includes reduction in alpha-SMA levels in comparison to untreated cells or cells treated with a reference peptide that has an amino acid sequence as denoted by SEQ ID NO: 3.

[0124] In some embodiments, all chiral amino acids (AAs) of the reference peptide are L-amino acids (all-lAAs). In some embodiments, the reference L-peptide has the amino acid sequence MEQGEFLNYD including all-lAAs.In some embodiments, the isolated D-peptide is capable of affecting neuroligin 4 (NLGN4) -Neurexin ip (Nrxi ) protein-protein interaction.

[0125] Reference is now made to FIG. 1 presenting D-peptide reduced activation of human hepatic stellate cells, as demonstrated in Example 1.

[0126] Moreover, the ability of the isolated D-peptide of SEQ ID NO: 2 to affect proliferation of cultured HSCs was evaluated in a dose dependent in the range of 250 nM - 8000 nM, and compared to un-treated HSCs.

[0127] As demonstrated herein, it was surprisingly found that, according to some embodiments, the interfering D-peptides of the invention can inhibit HSCs proliferation, as exemplified by MTS assay.

[0128] In some embodiments, the isolated D-peptide is capable of inhibiting Hepatic Stellate Cells (HSCs) proliferation and / or viability, in comparison to untreated cells or cells treated with a reference L-peptide that has an amino acid sequence as denoted by SEQ ID NO: 3. Each possibility is a separate embodiment.

[0129] Reference is now made to FIG. 2 presenting D-peptide inhibition of proliferation of human hepatic stellate cells, as demonstrated in Example 2.

[0130] The stability of the D-peptide was assessed in human serum, and compared to the stability of the L-peptide. The stability in human serum is indicative of the ability of the D-peptides to resist enzymatic or metabolic degradation.

[0131] As demonstrated herein, it was advantageously found that, according to some embodiments, the D-peptides of the invention has an improved stability in human serum in comparison to a reference L-peptide that has an amino acid sequence as denoted by SEQ ID NO: 3; wherein all chiral amino acids (AAs) of the reference peptide are L-amino acids (all-lAAs).

[0132] In some embodiments, the improved serum stability of the D-peptide includes increased resistance to protease degradation.

[0133] In some embodiments, the improved stability comprises at least 2-fold higher D-peptide concentration (nM) after incubation of a period between at least 10 minutes and 24 hours in human serum at a temperature of about 37°C, in comparison to the reference peptide denoted by SEQ ID NO: 3 incubated at identical conditions.In some embodiments, the improved stability comprises at least 3 -fold higher D-peptide concentration (nM) after incubation of a period between at least 1 hour and 24 hours in human serum at a temperature of about 37°C, in comparison to the reference peptide denoted by SEQ ID NO: 3 incubated at identical conditions.

[0134] In some embodiments, the improved stability comprises at least 20-fold higher D-peptide concentration (nM) after incubation of a period between at least 2 hours and 24 hours in human serum at a temperature of about 37°C, in comparison to the reference peptide denoted by SEQ ID NO: 3 incubated at identical conditions.

[0135] In some embodiments, the isolated D-peptide is stable for at least 120 minutes at 37°C in serum.

[0136] In some embodiments, the improved stability of the D-peptide comprises essentially constant peptide concentration (nM) during incubation in human serum for a period of up to 2-4 days or more, at a temperature of about 37°C, compared to an initial concentration of the D-peptide.

[0137] As used herein, the terms “essentially” and “substantially” are synonymous and when referring to a stated material such as a composition, a substance, and the like, is meant to encompass variations of in some embodiments, ±0.1%, or in some embodiments, ±1%, or in some embodiments, ±2%, or in some embodiments, ±5%, or in some embodiments, ±10%, or in some embodiments, ±20% from a stated amount, as such variations / deviations are appropriate to perform the disclosed methods.

[0138] In some embodiments, the essentially constant D-peptide concentration (nM) includes less than 20% reduction, or less than 15% reduction, or less than 10% reduction, or less than 5% reduction, or less than 1.0% reduction in the concentration of the D-peptide for a period of up to 2-4 days or more, at a temperature of about 37°C in human serum, compared to an initial concentration of the D-peptide. Each possibility is a separate embodiment.

[0139] Reference is now made to FIGs.3A-3B presenting D-peptide stability in human serum ex vivo, as detailed in Example 3.

[0140] Moreover, the D-peptide exhibits superior stability in human serum compared to the reference L-peptide, while retaining substantially steady concentrations of less than 20% decrease in its initial concentrations up to day 6 in human serum and less than50% decrease up to day 14 in human serum, suggesting it has a remarkable ability to withstand enzymatic degradation under these conditions.

[0141] In some embodiments, the concentration (nM) of the isolated D-peptide decreases by less than 50% after incubation of up to 10-12 days in human serum at a temperature of about 37°C, compared to an initial concentration of the D-peptide.

[0142] In some embodiments, the concentration (nM) of the isolated D-peptide decreases by less than 50% after incubation of up to 14 days in human serum at a temperature of about 37°C, compared to an initial concentration of the D-peptide.

[0143] Hence, in some embodiments, the half-life of the D-peptide 3 days or more, 7 days or more, 14 days or more, in human serum. In some embodiments, the isolated D-peptide has a half-life of at least 0.5 days, 1 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days ,9 days, 10 days, 11 days, 12 days, 13 days 14 days, or more in serum. Each possibility is a separate embodiment.

[0144] Reference is now made to FIG. 3C presenting D-peptide stability in human serum ex vivo, as demonstrated in Example 3.

[0145] The isolated D-peptide half-life was estimated in vivo in model mice for liver fibrosis, namely, CC14-induced acute hepatic fibrosis model mice.

[0146] As demonstrated herein, it was advantageously found that, according to some embodiments, the D-peptide of the invention has a half-life of approximately 1.5 hours following intravenous injection to CCh treated mice.

[0147] In some embodiments, the isolated D-peptide has a half-life of about 1.5 hours when intravenously injected into mice blood.

[0148] In some embodiments, the isolated D-peptide has a half-life of at least 90 minutes in vivo.

[0149] In some embodiments, the isolated D-peptide has a half-life of at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 60 minutes, at least about 70 minutes, at least about 80 minutes, at least about 90 minutes, or more in vivo. Each possibility is a separate embodiment.

[0150] Surprisingly, in some embodiments, the D-peptide could still be detected in the blood of the CCh treated mice after 15-60 minutes, or after 0.5, 1, 6, 12, 24, 48, or 72hours. In some embodiments, the isolated D-peptide is capable of deactivating HSCs, attenuating HSCs proliferation and / or affecting interactions between neuroligin 4 (NLGN4) and Neurexin ip (Nrxi ). Each possibility is a separate embodiment.

[0151] According to some embodiments, stimulation of NK cells with a D-peptide of the invention can elevate / increase / improve the activity of the NK cells.

[0152] According to some embodiments, stimulation of NK cells with increasing concentrations of a D-peptide can elevate / increase / improve the activity of the NK cells in a dose-dependent manner.

[0153] According to some embodiments, stimulation of NK cells with increasing concentrations of a D-peptide of the invention can increase the expression levels of CD107a in NK cells. In some embodiments, the increase is dose-dependent.

[0154] According to some embodiments, stimulation of NK cells with increasing concentrations of NLGN4-Nrxip PPI D-peptide increases the levels of CD107a, in an inverse dose-dependent pattern. In some embodiments, the inverse dose-dependent pattern is that the levels of CD 107a decrease at higher D-peptide concentration.

[0155] According to some embodiments, stimulation of hepatic stellate cells (HSCs) with increasing concentrations of NLGN4-Nrxi PPI D-peptide deactivates the cells.

[0156] According to some embodiments, stimulation of hepatic stellate cells (HSCs) with increasing concentrations ofNLGN4-Nrxip PPI D-peptide decreases the levels of aSMA (Alpha Smooth Muscle Actin).

[0157] According to some embodiments, the present invention provides methods and compositions for treating, attenuating, and / or preventing progression of a liver disorder by activating natural killer cells (NK cells) and synergistically deactivating hepatic stellate cells (HSCs) by utilizing the isolated D-peptides of the invention.

[0158] According to some embodiments, the D-peptide is capable of mediating deactivation of HSCs, in particular, more prominently in the presence of activated NK cells.

[0159] According to some embodiments, pre-activation of NK cells with the D-peptide(s) can render the cells more functional in deactivating HSCs cells.According to some embodiments, pre-activation of NK cells with the D-peptides of the invention can synergistically deactivate HSCs, as indicated, for example, by profound reduction in aS MA levels.

[0160] According to some embodiments, the effect of pre-activation of NK cells deactivating HSCs is dose-dependent. The term "activated NK cells" relates to NK cells treated (administered, expressing, introduced with) an isolated D-peptide of the invention, or a composition including the same.

[0161] According to some embodiments, NLGN4-Nrxip D-peptide can stimulate NK cells activity and / or cause reduction in the activity of HSCs in monocultures. Each possibility is a separate embodiment.

[0162] According to some embodiments, D-peptide-activated NK cells can synergistically function to downregulate / deactivate HSCs. According to some embodiments, the synergistic downregulation and / or deactivation of HSCs is represented by reduction in aSMA levels of HSCs co-cultured with NK cells prestimulated with NLGN4-Nrxip PPI interfering D-peptide. The reduction is compared to HSCs in monoculture stimulated NLGN4-Nrxip PPI interfering D-peptide or to coculture of unstimulated NK and HSCs.

[0163] According to some embodiments, following co-culturing with pre-activated-NK cells, two populations of HSCs may be identified, said populations characterized according to the aSMA expression levels, being medium or high (relatively).

[0164] According to some embodiments, NLGN4-Nrxip PPI interfering D-peptides promote deactivation of HSCs, much more profoundly when first utilized to prestimulate NK cells killing activity.

[0165] According to some embodiments, there is provided a method of promoting anti-fibrotic effects in the liver, the method includes administering any isolated D-peptide of the invention, or compositions including the same, to a subject in need thereof.

[0166] According to some embodiments, NLGN4-Nrxip PPI interfering D-peptide can synergistically deactivate co-cultured HSCs cells through activated NK cells.

[0167] According to some embodiments, the NLGN4-Nrxip interfering D-peptides can advantageously promote anti-fibrotic effects, by modulating NLGN4X / p-neurexinaxis through functionally reducing hepatic stellate cell-mediated phagocytosis and / or increasing NK cells adherence-mediated killing of hepatic stellate cells. Each possibility is a separate embodiment.

[0168] According to some embodiments, the D-peptides may disrupt the NLGN4X / P-neurexin axis by affecting different aspects of the HSCs / NK intercellular interaction such as, but not limited to: the ratio between different subpopulations of adhered cells expressing the NK cell-specific marker CD56+ and the HSCs activation marker aSMA, as either double-positive CD56+ / aSMA+ or single positive CD56+ / aSMA-. Each possibility is a separate embodiment.

[0169] According to some embodiments the D-peptide may promote reduction of a subpopulation expressing CD56+ / aSMA+ double-positive markers, that represent a subpopulation of NK cells that adhere to active HSCs, as were evaluated following the NK killing. In some embodiments, the effect of the D-peptide is dose dependent.

[0170] In some embodiments, NK cells activated (treated) by the D-peptides may be less engulfed into HSCs, compared to untreated HSCs / NK cells. According to some embodiments, D-peptide-activated NK cells may be more functional in killing HSCs, compared to untreated HSCs / NK cells.

[0171] According to some embodiments, administration of a D-peptide may promote increase in a subpopulation of NK cells expressing CD56+ / aSMA- single-positive markers that represents aNK cell subpopulation that adheres to inactive HSCs cells. In some embodiments, the effect of the D-peptide is dose dependent.

[0172] According to some embodiments, there is provided a method of activating NK cells to thereby increase their affinity / binding / attachment / adherence to HSCs, the method including providing (administering) the cells with any of the D-peptides of the invention, or compositions including the same.

[0173] In some embodiments, D-peptide-activated NK cells are more capable of attaching to inactive HSCs, relative to untreated HSCs / NK cells. In some embodiments, D-peptide-activated NK cells are more capable of attaching to active HSCs, to thereby deactivate HSCs, relative to untreated HSCs / NK cells interaction. In some embodiments, inactive HSCs cells are less functional in engulfing and killing active-NK cells relative to inactive NK cells.According to some embodiments, the D-peptide of the invention can advantageously promote reduction in subpopulation of cells expressing CD56+ / aSMA+ double-positive markers and increase in subpopulation expressing CD56+ / aSMA- single-positive markers, relative to untreated HSCs / NK cells. In some embodiments, the effect is dose dependent.

[0174] According to some embodiments, NLGN4-Nrxip PPI interfering D-peptides can modulate NLGN4X / p-neurexin axis by functionally reducing hepatic stellate cell activation and / or increasing NK cells adherence-mediated killing of hepatic stellate cells. Each possibility is a separate embodiment.

[0175] According to some embodiments, there is provide a method for enhancing killing / inactivation of HSC cells and / or enhancing antifibrotic effects, the method includes activating NK cells with the D-peptides of the invention, or a composition including the same. In some embodiments, the activation is performed in-vivo. In some embodiments, the activation may be performed in-vitro and the activated NK cells may be administered (systemically and / or locally) to the subject.

[0176] According to some embodiments, D-peptides-activated NK cells exhibit improved adherence to HSCs, thereby potentiating enhanced anti-fibrotic effects and / or killing of HSCs.

[0177] In some embodiments, D-peptide-activated NK cells can cause HSCs to perform less phagocytosis ofNK cells. In some embodiments, D-peptide-activated NK cells may cause HSCs to be less functional (reduced functionality) in killing NK cells.

[0178] According to some embodiments, the D-peptide can modulate / disrupt / block the NLGn4X / p-neurexin axis, by reducing, inhibiting, or preventing immune synapse between HSCs and NK cells that attenuates NK cell function.

[0179] According to some embodiments, there is provided a method of inhibiting or ameliorating liver fibrosis in a subject in the need thereof, the method includes administrating a D-peptide of the invention, or a composition including the same to a subject in need thereof. According to some embodiments, the D-peptide exhibit reduced to minimal side effects, such as, for example, liver toxicity.

[0180] According to some embodiments, the D-peptide of the invention can inhibit liver fibrosis in vivo.According to some embodiments, the interfering D-peptide can be used to ameliorate several pathological characteristics evident through examination of the sera (serum) and liver biopsies of fibrotic liver. In some embodiments, the pathological characteristics may be selected from, but not limited to: serum levels of ALT, fibrotic liver inflammation, swelling levels of centrilobular hepatocytes, necrotic areas of high infiltrating inflammatory cells with steatosis, micro- and macrovascular steatosis, collagen deposition in perisinusoidal areas and / or the fibrous dense tissue, accumulation of thick fibrotic tissue, mRNA and / or protein levels of fibrotic biomarkers expressed in the liver, including aSMA, collagen I, CREBP, MMP-9, and the like, or any combination thereof. Each possibility is a separate embodiment. In some embodiments, the D-peptide can reduce levels of serum alanine transaminase (ALT).

[0181] According to some embodiments, the D-peptide can ameliorate and / or prevent fibrotic liver inflammation. Each possibility is a separate embodiment. According to some embodiments, the D-peptide can prevent and / or reduce swelling of centrilobular hepatocytes and reduce necrotic areas of high infiltrating inflammatory cells with steatosis in fibrotic livers. Each possibility is a separate embodiment.

[0182] According to some embodiments, the D-peptide can reverse histological alterations found in fibrotic livers. According to some embodiments, the D-peptide can prevent and / or reduce micro- and macrovascular steatosis in fibrotic livers. Each possibility is a separate embodiment.

[0183] According to some embodiments, the D-peptide can prevent and / or reduce collagen deposition in perisinusoidal areas and / or the fibrous dense tissue in fibrotic livers. Each possibility is a separate embodiment. According to some embodiments, the D-peptide can prevent and / or reduce accumulation of thick fibrotic tissue in fibrotic livers. Each possibility is a separate embodiment.

[0184] According to some embodiments, D-peptide may prevent and / or reduce mRNA and / or protein levels of fibrotic biomarkers expressed in the liver, such as, but not limited to aS MA, collagen I, CREBP, and MMP-9. Each possibility is a separate embodiment.

[0185] According to some embodiments, the NLGN4-Nrxip PPI D-peptide can inhibit liver fibrosis in in vivo.According to some embodiments the D-peptide may modulate P-neurexin expression and / or activity in HSCs and / or F-actin expression of liver NK cells. Each possibility is a separate embodiment. According to some embodiments, the D-peptide can prevent or reduce P-neurexin expression and / or activity in fibrotic livers. Each possibility is a separate embodiment.

[0186] According to some embodiments, the D-peptide can increase F-actin expression in fibrotic livers. According to some embodiments, the D-peptide can increase F-actin expression in fibrotic livers.

[0187] According to some embodiments, the D-peptide can deactivate HSCs through inhibition of P-neurexin expression or activity.

[0188] According to some embodiments, the D-peptide can increase F-actin expression in NK cells as evident by its co-localization with Natural Cytotoxicity Receptors (NKP46) inside NK cells. According to some embodiments, the D-peptide can restore NK activity through increased expression of F-actin, which is necessary for normal cellular function and motility. According to some embodiments, the NLGN4-Nrxip PPI D-peptide can modulate P-neurexin expression of HSCs and / or F-actin expression of liver NK cells. Each possibility is a separate embodiment.

[0189] According to some embodiments, there is provided a host cell comprising the isolated D-peptide(s) of the invention. In some embodiments, the host cell may be administered with the isolated D-peptide(s). In some embodiments, the host cell is NK cell.

[0190] Accordingly, in some embodiments, the D-peptide, or a composition comprising the same may be used for treatment of liver disorders and / or related cancer therapy.

[0191] According to some embodiments, the liver disorder may be selected from: fibrosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, hepatitis, viral hepatitis, liver adenoma, insulin hypersensitivity, liver cancer, hepatocellular carcinoma, liver metastasis, and the like, or any combination thereof. Each possibility is a separate embodiment.According to some embodiments, the liver disorder is fibrosis. According to some embodiments, the liver disorder is non-alcoholic fatty liver disease (NAFLD). According to some embodiments, the liver disorder is non-alcoholic steatohepatitis (NASH). According to some embodiments, the liver disorder is cirrhosis. According to some embodiments, the liver disorder is hepatitis. According to some embodiments, the liver disorder is liver adenoma. According to some embodiments, the liver disorder is insulin hypersensitivity. According to some embodiments, the liver disorder is liver cancer. According to some embodiments, the liver disorder is hepatocellular carcinoma. According to some embodiments, the liver disorder is a combination of diseases.

[0192] According to some embodiments, any suitable route of administration to a subject may be used for the isolated D-peptides or the compositions of the present invention, including but not limited to, local and systemic routes. Each possibility is a separate embodiment. In some embodiments, exemplary suitable routes of administration include, but are not limited to: oral, intra-nasal, parenteral, intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intracerebroventricular, intrastriatal, intracranial and into the spinal cord, intraductal, sublingual, rectal, transdermal, vaginal, topical, by enema or by inhalation. Each possibility is a separate embodiment. According to another embodiment, systemic administration of the composition is via an injection. For administration via injection, the composition may be formulated in an aqueous solution, for example in a physiologically compatible buffer including, but not limited, to Hank’s solution, Ringer’s solution, or physiological salt buffer. Formulations for injection may be presented in unit dosage forms, for example, in ampoules, or in multi-dose containers with, optionally, an added preservative.

[0193] According to another embodiment, systemic administration is through a parenteral route. According to some embodiments, parenteral administration may be intravenous, intra-arterial, intramuscular (i.m.), intraperitoneal (i.p.), intradermal, or subcutaneous (s.c). Each of the abovementioned administration routes represents a separate embodiment of the present invention.

[0194] In some embodiments, the administration is by subcutaneous (s.c.) administration.According to another embodiment, parenteral administration may be performed by bolus injection or continuous infusion. Each possibility is a separate embodiment.

[0195] According to some embodiments, preparations of the composition of the invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions, each representing a separate embodiment of the present invention.

[0196] According to some embodiments, the administration may include any suitable administration regime, depending, inter aha, on the medical condition, patient characteristics, administration route, and the like. In some embodiments, administration may include administration twice daily, every day, every other day, every third day, every fourth day, every fifth day, once a week, once every second week, once every third week, once every month, and the like. Each possibility is a separate embodiment.

[0197] According to some embodiments, the D-peptides or the pharmaceutical composition including the same may be administered at a dose of from about 1 mg / kg to about 200 mg / kg. In some embodiments the D-peptides or the pharmaceutical composition may be administered at a dose of from about 25 mg / kg to about 150 mg / kg. In some embodiments the D-peptides or the pharmaceutical composition may be administered at a dose of from about 50 mg / kg to about 100 mg / kg. In some embodiments the D-peptides or the pharmaceutical composition may be administered at a dose of from about 60 mg / kg to about 80 mg / kg.

[0198] According to some embodiments, the administration may be performed once, 1-3 times per day, 1-7 times per week, 1-4 times per months, or any combination thereof.

[0199] According to some embodiments, there are provided kits comprising the isolated D-peptides or composition comprising the same, as disclosed herein. In some embodiments, such kits / compositions can be used, for example, in the treatment of various liver-related conditions, such fibrosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, hepatitis, viral hepatitis, liver adenoma, insulin hypersensitivity, liver cancer, hepatocellular carcinoma, liver metastasis. Each possibility is a separate embodiment. In some embodiments, the kit may further include one or more reagents, buffers and / or instructions for using thesame. In some embodiments, the composition may further include a pharmaceutically acceptable carrier.

[0200] According to some embodiments, there are provided methods and compositions for treating, attenuating, and / or preventing progression of a liver cancer by inhibiting proliferation and / or attenuating oncogenicity and / or increasing programmed cell death of liver cancer cells by utilizing the isolated D-peptides of the invention. Each possibility is a separate embodiment.

[0201] According to some embodiments, there is provided a method of promoting anti-cancerous effects in the liver, the method including administering any isolated D-peptide of the invention, or compositions including the same, to a subject in need thereof, wherein the anti-cancerous effect comprises inhibiting proliferation and / or attenuating oncogenicity and / or increasing programmed cell death and / or reducing necrosis of liver cancer cells. Each possibility is a separate embodiment.

[0202] According to some embodiments, the D-peptide of the invention can inhibit / attenuate proliferation and / or oncogenicity of liver cancer cells. Each possibility is a separate embodiment.

[0203] According to some embodiments, the D-peptide of the invention may inhibit cell division of cancer cells by attenuating entry of the cancer cells into a state of S-phase or G2-M-phase. According to some embodiments, the D-peptide of the invention shifts cancer cells from existing at a state of S-phase or G2-M phase towards existing at a state of G1 phase. Each possibility is a separate embodiment. According to some embodiments, the D-peptide of the invention inhibits cell division of liver cancer cells by attenuating entry of the cancer cells into a state of S-phase or G2-M-phase. Each possibility is a separate embodiment. According to some embodiments, the D-peptide of the invention shifts liver cancer cells from a state of S-phase or G2 / M-phase towards G1 phase. According to some embodiments, the inhibiting / attenuating proliferation and / or oncogenicity of liver cancer cells using the D-peptide of the invention is associated with attenuating entry of the cancer cells into a state of S-phase or G2-M-phase and increasing entry of the cancer cells into existing in a state of G1 phase.

[0204] According to some embodiments, the NLGN4-Nrxip PPI D-peptide can modulate proliferation of Hepatocellular carcinoma (HCC) cells in vitro and / or in-vivo.According to some embodiments, the inhibiting / attenuating proliferation and / or oncogenicity of liver cancer cells using the D-peptide of the invention may be associated with reduces activation of Akt-signaling pathway and / or mTOR-signaling pathway. Each possibility is a separate embodiment. According to some embodiments, the D-peptide of the invention reduces activation of Akt-signaling pathway and / or mTOR-signaling pathway in cancer cells, thereby inhibiting proliferation and / or oncogenicity. Each possibility is a separate embodiment. According to some embodiments, the D-peptide of the invention reduces activation of Akt-signaling pathway and / or mTOR-signaling pathway in liver cancer cells (HCC), thereby inhibiting proliferation and / or oncogenicity. Each possibility is a separate embodiment. According to some embodiments, the D-peptide of the invention reduces the level of phosphorylated AKT / mTOR / P70S6K

[0205] According to some embodiments, the isolated D-peptide or the composition including the same, may be used for in attenuating proliferation and / or oncogenicity of liver cancer cells. Each possibility is a separate embodiment.

[0206] According to some embodiments, the isolated D-peptide or the composition including the same, may be used for in attenuating proliferation and / or oncogenicity of liver cancer cells is associated with an increase in programmed cell death / apoptosis of the cancer cells and / or reduction in necrosis of the cancer cells. Each possibility is a separate embodiment.

[0207] According to some embodiments, the NLGN4-Nrxip PPI D-peptide can modulate cellular proliferation and / or oncogenic signaling of Hepatocellular carcinoma (HCC) cells in vitro or in-vivo. Each possibility is a separate embodiment.

[0208] According to some embodiments, the D-peptide of the invention may increase programmed cell death of cancer cells. According to some embodiments, the D-peptide of the invention may reduce necrosis of cancer cells. According to some embodiments, the D-peptide of the invention may increase programmed cell death of liver cancer cells (HCC) .According to some embodiments, the D-peptide of the invention may reduce necrosis of liver cancer cells (HCC).

[0209] According to some embodiments, the inhibiting / attenuating proliferation and / or oncogenicity of liver cancer cells using the D-peptide of the invention may beassociated with increased programmed cell death or reduced necrosis of liver cancer cells. Each possibility is a separate embodiment.

[0210] According to some embodiments, the NLGN4-Nrxip PPI D-peptide can modulate cell death of Hepatocellular carcinoma (HCC) cells in vitro or in-vivo.

[0211] According to some embodiments, the NLGN4-Nrxip PPI D-peptide may have an inhibitory effect on various types of cancer cells.

[0212] According to some embodiments, the present invention stems, in part, from the surprising finding that specific isolated D-peptides are capable of deactivating HSCs or attenuating HSCs proliferation or affecting (for example, by interfering or inhibiting) NLGN4X-Nrxip protein-protein interactions in a manner that crucially disrupts functional intercellular attributes of, for example, hepatic satellite cells (HSCs) or liver cancer cells, which play an essential role in the progression of various liver disorders. Each possibility is a separate embodiment.

[0213] According to some embodiments, without wishing to be bound by any theory or mechanism, pre-activation of NK cells with D-peptides of the invention, can synergistically deactivate HSCs, thereby treating or ameliorating related liver conditions.

[0214] According to some embodiments, without wishing to be bound by any theory or mechanism, hepatocellular cells (HCC) treated with the isolated D-peptides disclosed herein, proliferate / divide at slower rate and / or more prone for programed cell death / apoptosis. Each possibility is a separate embodiment. Accordingly, the isolated D-peptides disclosed herein can be used for treating or ameliorating liver related conditions, such as, liver cancer.

[0215] According to further embodiments, without wishing to be bound by any theory or mechanism, the NLGN4-Nrxip interfering isolated D-peptides can advantageously promote anti-fibrotic effects, in some embodiments by functionally reducing hepatic stellate cell activation-mediated phagocytosis and / or increasing NK cells adherence-mediated killing of hepatic stellate cells (HSCs). Each possibility is a separate embodiment.

[0216] According to some embodiments, there is provided a composition which includes the isolated D-peptide(s), and a pharmaceutically acceptable carrier.According to some embodiments, the composition may include a plurality of isolated D-peptides.

[0217] According to some embodiments, the isolated D-peptide(s), or the composition including the same may be used for treating, attenuating, and / or preventing progression of a liver disorder, in a subject in need thereof.

[0218] According to some embodiments, the liver disorder may be selected from: fibrosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, hepatitis, viral hepatitis, liver adenoma, insulin hypersensitivity, liver cancer, liver metastasis or any combination thereof. According to some embodiments, the liver disorder is liver fibrosis or liver cancer. Each possibility is a separate embodiment.

[0219] According to some embodiments, the isolated D-peptide(s), or the composition including the same may be used for treating, attenuating, and / or preventing progression of a cancer condition in a subject in need thereof. In some embodiments, the cancer may be selected from: liver cancer, cholangiocarcinoma, liver metastasis, colorectal cancer, breast cancer and prostate cancer. Each possibility is a separate embodiment.

[0220] According to some embodiments, reducing activation of hepatic stellate cells (HSCs) comprises increased susceptibility of killing thereof by activated natural killer (NK) cells and / or reduced engulfment by activated NK cells. Each possibility is a separate embodiment.

[0221] According to some embodiments, reducing activation of hepatic stellate cells (HSCs) comprises increased susceptibility for killing thereof by natural killer (NK) cells.

[0222] According to some embodiments, the administration may include systemic or localized administration. According to some embodiments, the administration may include systemic, sub-cutaneous administration. According to some embodiments, the method may further include administering at least one additional therapeutic agent.

[0223] According to some embodiments, there is provided a natural killer (NK) cell including the isolated D-peptide or composition including the same. Each possibility is a separate embodiment. According to some embodiments, the isolated D-peptide is introduced or penetrates the NK cell as a peptide. According to some embodiments,the NK cell is an activated cell capable of reducing or inhibiting activity of hepatic stellate cells.

[0224] According to some embodiments, there is provided a composition including the NK cell comprising / harboring the isolated D-peptide or composition including the same. Each possibility is a separate embodiment.

[0225] According to some embodiments, the NK cell including the isolated D-peptide or composition including the same may be used for treating, attenuating and / or preventing progression of a liver disorder in a subject in need thereof.

[0226] According to some embodiments, the NK cell including the isolated D-peptide or composition including the same may be used for treating, attenuating and / or preventing progression of cancer condition in a subject in need thereof.

[0227] According to some embodiments, the NK cells including the isolated D-peptide or composition including the same may be administered systemically. According to some embodiments, the NK cells including the isolated D-peptide or composition including the same may be administered in combination with at least one additional therapeutic agent.

[0228] According to some embodiments, reduction in activity of HSC by NK-cells comprising the isolated D-peptide, or the composition comprising the same, is synergistic, with respect to reduction in activity of HSC by NK-cells not comprising the isolated D-peptide, and with respect to reduction in activity of HSC by the isolated D-peptide when not in the presence of D-peptide activated NK cells . Each possibility is a separate embodiment.

[0229] According to some embodiments, the NK-cells including the isolated D-peptide or the composition comprising the same, may have reduced susceptibility of being engulfed into HSCs, and / or increased functionality in killing HSCs, and / or increased attachment / adherence to HSCs. Each possibility is a separate embodiment.

[0230] According to some embodiments, there is provided a method of treating, attenuating and / or preventing progression of a liver disorder in a subject in need thereof, the method includes administering a therapeutically effective amount of the D-peptide or a composition including the same.According to some embodiments, there is provided a method of treating, attenuating and / or preventing progression of a liver disorder in a subject in need thereof, the method includes administering a therapeutically effective amount of the NK cell including the D-peptide and / or the composition including the NK cell. Each possibility is a separate embodiment.

[0231] According to some embodiments, there is provided a method of treating, attenuating and / or preventing progression of cancer condition in a subject in need thereof, the method includes administering a therapeutically effective amount of one or more of: an NK cell comprising the D-peptide, composition including the NK cell, the D-peptide(s), and / or composition including the D-peptide(s).

[0232] The following examples are presented to provide a more complete understanding of the invention. The specific techniques, conditions, materials, proportions and reported data set forth to illustrate the principles of the invention are exemplary and should not be construed as limiting the scope of the invention.

[0233] EXAMPLES:

[0234] Materials and Methods:

[0235] D-enantiomer peptide synthesis:

[0236] H-(dM)(dE)(dQ)G(dE)(dF)(dL)(dN)(dY)(dD)-OH (SEQ ID NO: 2)

[0237] The D-peptide of SEQ ID NO: 2, having 9 chiral amino acids (AAs) synthesized as D-amino acid (all-dAAs) was synthesized using standard Fmoc-based solid phase peptide synthesis on a CEM Liberty Blue synthesizer using Wang resin. Single coupling of amino acids was carried out at 90°C using a 5 -fold excess and DIC / Oxyma activation for two min. Fmoc deprotection was performed at 90°C using 20% piperidine in DMF for one min. After completing the synthesis, the resin-bound peptide was cleaved and deprotected with 10 ml of TFA / water / thioanisole / triisopropylsilane (86:5:5:2 ratio) at room temperature for 3 hours. The cleaved peptide was precipitated in cold diethyl ether, and the pellet obtained by centrifugation (3600 ppm) was reconstituted in a 1:1 water / acetonitrile containing 0.1% TFA, then lyophilized. The lyophilized crude peptide was purified by RP-HPLC (Cl 8, 10pm, 25 x 250mm column), using a gradient of 12-27% Buffer B over 30 minutes (Buffer A= 0.1% TFA in water, Buffer B = 0.08% TFA in acetonitrile) at a flow rate of 30 ml / min with UV detection of 220 nm. Thepeptide was analyzed, and pure fractions were pooled and lyophilized. Analytical LC / MS data was obtained on an analytical column (C 18, 1.7pm, 130 A, 2.1 x 50mm) using a gradient of 1-55 Buffer B over 2 minutes (Buffer A = 0.05% TFA in water, Buffer B = 0.05% TFA in acetonitrile) at a flow rate of 0.8 ml / min at 50 °C with UV detection of 215 nm and 220 nm.

[0238] Cell culture experiments:

[0239] Peptide synthesis: D-peptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, and reference L-peptide of SEQ ID NO: 3 are synthesized by solid phase peptide synthesis

[0240] Cells: primary NK cells are isolated from patients having advanced fibrosis, human hepatic stellate cells (LX2) are commercially purchased.

[0241] Cell viability assay: In experiments involving NK cells, cell viability is assessed by propidium iodide exclusion.

[0242] Monoculture conditions: isolated peptides are supplemented to media of cultured NK cells for 6h stimulation.

[0243] Co-culture conditions: co-cultures conditions - peptides are supplemented to media of cultured NK cells for 6h stimulation followed by a wash, trypsinization, and coculturing on LX2 cells for an additional 24h.

[0244] Fluorescence-activated cell sorting (FACS): antibodies against aSMA, CD 107, CD56. After analysis using LSR Fortessa analyzer, raw data is transferred to analysis program such as FCS Express v7.0.

[0245] Gene expression analysis by Flow cytometry: detection of proliferative or oncogenic markers expressed in Hep3B HCC cells, is performed according to a standard protocol, using antibodies for a-feto-protein (a-FP), Carboxy Fluorescein Succinimidyl Ester (CSFE), PDGFRA and MKI67 and of phosphorylated AKT / mTOR / P70S6K.

[0246] Cell cycle analysis by Flow cytometry: evaluation of cell cycle stage of Hep3B HCC cells, is performed according to a standard protocol, using Propidium-Iodide (PI)-staining of nuclei.Cell death assessment by flow cytometry: evaluation of cell viability, necrosis and apoptosis of Hep3B HCC cells is performed according to a standard protocol, using Propidium-Iodide (Pl)-staining of nuclei, phosphatidylserine staining of annexin V and CDKN2A.

[0247] Western Blot analysis: total protein extraction from of Hep3B HCC cells and WB analysis for P-neurexin are performed according to a standard protocol, using anti-human- -neurexin and anti-GAPDH antibodies. Anti-Neurexin-l-Beta (dilution : 1 :1000) was purchased from Antibodies incorporated (Davis, CA, USA), a-Smooth Muscle Actin (dilution : 1 :1000), Lysosome-associated membrane protein 1 (dilution : 1 :1000), GAPDHmAb (dilution : 1 : 5000), Anti-rabbit IgG, HRP -linked Antibody (dilution : 1 : 5000) & Anti -mouse IgG, HRP -linked Antibody (dilution : 1 : 5000), was purchased from Cell Signalling Technology (Danvers, MA, USA).

[0248] In-vivo experiments:

[0249] Animal model: WT naive C57BL / 6J male mice (12 weeks) are either induced for acute hepatic fibrosis by i.p injections of 0.5pl pure carbon tetrachloride CCh / g body weight 3X / week for four weeks as an advanced model of hepatic fibrosis or left untreated. NLGN4-Nrxip PPI inhibitor D-peptide is i.p or s.c. injected to CCI4 induced mice as preventive doses from the beginning of the model, starting at 1st week of CCI4 at a concentration of 5 and 10 mg / mice 3X / week for four weeks (5mg / mice and lOmg / mice). A reference L-peptide was also i.p or s.c. injected to CCI4 induced mice (5mg / mice and lOmg / mice). Food and water intake are recorded every week. Mice are sacrificed two days after the final CCI4 injection. To this end, the animals are weighed and anesthetized before cervical dislocation.

[0250] In some experiments, D-peptide (e.g., having SEQ ID NO: 2, “EUJI-4”) was administered by subcutaneous or intraperitoneal route three times a week in the morning for two weeks. Rezdiffra (Resmetirom) was administered by oral route once daily in the morning for two weeks. Treatment was initiated post CCI4 model development (Day-28 of the study initiation) as therapeutic mode.

[0251] Histological and biochemical assessment of mice livers: The posterior one-third of the liver is fixed in 4% formalin for 24 hours and then paraffin-embedded in an automated tissue processor. Sections (7 mm) are stained for H&E for evaluatingsteatosis, necro-inflammatory regions and apoptotic bodies, 0.1% Sirius red F3B in saturated picric acid as well as Masson’s trichrome stain for connective tissue. Mice whole blood samples are collected at the sacrificing day, centrifuged at 3500 rpm for 10 minutes at 4°C. Serum ALT concentrations are determined using ELISA.

[0252] RNA isolation. cDNA preparation and real-time PCR: Total cellular RNA is isolated from liver tissue and cDNA generated, is . Real time PCR is performed for quantification of aSMA, Collagen I and Cyclic Adenosine Monophosphate-Responsive Element Binding Protein (CREBP) gene expressions, which are normalized to the expression a housekeeping gene.

[0253] Western blot analysis: Liver proteins are extracted, and immunoblotting is performed. Blots are incubated with anti-human / mice aSMA, anti-human-p-Nrxn, Lysosome-associated membrane protein 1, anti-mice F-actin, anti-GDPH and / or anti-human / mice MMP-9.

[0254] Anti -Neurexin-1 -Beta (dilution : 1 :1000) was purchased from Antibodies incorporated (Davis, CA, USA), a-Smooth Muscle Actin (dilution : 1 :1000), Lysosome-associated membrane protein 1 (dilution : 1 :1000), GAPDHmAb (dilution : 1 : 5000), Anti-rabbit IgG, HRP-linked Antibody (dilution : 1 : 5000) & Anti-mouse IgG, HRP-linked Antibody (dilution : 1 : 5000), was purchased from Cell Signalling Technology (Danvers, MA, USA).

[0255] To determine the amount of a-Smooth Muscle Actin, Lysosome-associated membrane protein 1 and Anti-Neurexin-l-Beta, western blotting analysis was performed. Liver lysates were centrifuged at 12,000 g at 4 °C for 15 min, and the supernatants were stored at -80 °C until required for analysis. Protein concentrations were measured using a protein assay kit (Bio-Rad, Hercules, CA, USA). Aliquots of the lysates (50 pg of protein) were applied to an 8 % sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred on to a polyvinylidene fluoride (PVDF) membrane. After blocking nonspecific sites with 5% BSA in Phosphate buffered saline with 0.1% Tween-20 (PBST), the membranes were subsequently incubated with specific primary antibodies a-Smooth Muscle Actin mouse mAb, Lysosome-associated membrane protein 1 Rabbit mAb, Anti-Neurexin-l-Beta mouse mAb, GAPDH Rabbit mAb for overnight at 40C. The membranes were subsequently incubated for 2 h with peroxideconjugated secondary antibodies (anti-rabbit Ig G-HRP and anti-mouse Ig G-HRP1:5000). Cell Signaling Technology (Danvers, MA, USA). The immunoreactive proteins were detected using an enhanced chemiluminescence (ECL) western blotting detection kit (Amersham Pharmacia Biotech, NY, USA). GAPDH was used as the standard for normalizing protein samples. The band intensities were quantified using a ImageJ (National Institutes of Health, Wayne Rasband).

[0256] Immunofluorescence staining: paraffin-embedded sections are deparaffinization, incubated in xylene and antigen retrieval is performed. Samples are than incubated overnight at 4°C with anti -mice F-actin and anti-mouse NKP46. Following washing with PBS, secondary antibodies are added for one hour at room temperature, and image capture is performed.

[0257] Fluorescence activated cell sorting (FACS): Mice liver NK cells are characterized as NK1.1. For assessment of NK activations, CD 107a; LAMP1 (lysosomal-associated membrane protein-1) markers are used. An isotype IgG labeled with the relevant fluorochrome is used as a control. Stained cells are analyzed with a flow cytometer.

[0258] Statistical Analysis: Statistical differences are analyzed with two-tailed unpaired Student's either t-test (for comparison between two groups) or one-way analysis of variance (one-way ANOVA with Newman-Keuls post-tests among multiple groups).

[0259] Example 1: D-peptide Deactivates Human Hepatic Stellate LX2 Cells Replacing the identity of amino acid (AA) in any peptide amino acid sequence can alter the structure of the peptide and disrupt its function

[0260] Even replacement of just a single chiral amino acid (AA) in the amino acid sequence MEQGEFLNYD with its un-naturally occurring D-enantiomeric AA is expected to result in an altered structure of the peptide that will negatively affect its activity and impair its function.

[0261] Here, the activity of the isolated D-peptide was first evaluated in cultured LX2 cells.The isolated D-peptide has an amino acid sequence as denoted by SEQ ID NO: 2, including 9 chiral amino acids (AAs) synthesized as D-amino acid: [H-( dM )( dE ) ( dQ ) G ( dE ) ( dF ) ( dL ) ( dN ) ( dY )( dD )-OH], the D-peptide of SEQ ID NO: 2 is also herein referred as all-dAAs D-peptide (or Luji-4).

[0262] The activity of the isolated D-peptide was compared to an isolated L-peptide (reference peptide) that has a corresponding amino acid sequence as denoted by SEQ ID NO: 3 including 9 chiral amino acids (AAs) synthesized as L-amino acids (all-lAAs): [MEQGEFLNYD] . The reference L-peptide of SEQ ID NO: 3 is also herein referred as all-lAAs L-peptide.

[0263] Additionally, the activity of each of the isolated D-peptide and the reference isolated L-peptide was compared to that of Rczdiffra™ (MGL) either alone, or in combination, and further compared to that of un-treated cells.

[0264] Methods - Briefly, LX2 cells were cultured on 12-well culture plates with DMEM+10% FBS overnight, after cells reaching confocal state D-peptide, L-peptide and MGL were added for 24-hours at 8 pM, 8 pM and 6 nM, respectively. Cells were then harvested and fixed using PFA, then permeabilization was performed using PBS + 0.1% Triton X-100, and antibody against a-SMA was then added, and targeted for detection and evaluation using FACS analyzer.

[0265] Results - As can be seen in FIG. 1, the isolated D-peptide (‘D-peptide’) deactivated LX2 cells as evident by the approximately 30%-40% reduction observed in a-SMA levels in comparison to untreated cells (‘not treated’) and by the observed approximately 15%-25% reduction in a-SMA levels in comparison to cells treated with the reference L-peptide (‘L-peptide’).

[0266] The isolated D-peptide exhibited better activity in deactivating LX2 cells also with respect to MGL (‘MGL’), as evident by the approximately 25%-35% reduction observed in a-SMA levels, as well as a better activity than MGL in combination with the reference L-peptide (‘MGL+L-Peptide’) as evident by the observed approximately 20%-30% reduction in a-SMA levels.

[0267] Furthermore, a small but statistically insignificant change of approximately 10%- 15% was observed in a-SMA levels between LX2 cells treated with the isolated D-peptide (‘D-peptide’) in comparison to treatment with the isolated D-peptide in combination with MGL(‘MGL+D-Peptide’).To conclude, the isolated D-peptide of SEQ ID NO: 2 showed the highest ability to deactivate human hepatic stellate cells, exhibiting better inhibitory effect than each of the reference L-Peptide or Rezdiffra™ (MGL) alone, or combined.

[0268] This is particularly advantageous and surprising since the effect of replacing L-amino acids with their corresponding D enantiomers in the sequence MEQGEFLNYD was unknown and thus expected to impair the function of the peptide. A corruption of activity would have been expected even for a replacement of a single chiral amino acids (AAs) in the sequence (for example, as denoted by the amino acid sequence of SEQ ID NO: 1), let alone for a replacement of all 9 chiral AAs as denoted by the amino acid sequence of SEQ ID NO: 2.

[0269] Example 2: D-peptide Inhibits Proliferation of Human Hepatic Stellate LX2 Cells Next, the ability of the isolated D-peptide of SEQ ID NO: 2 to affect proliferation of cultured LX2 cells was evaluated in a dose dependent manner in the range of 250 nM - 8000 nM, and compared to untreated LX2 cells.

[0270] Methods - Briefly, LX2 cells were cultured on 12-well culture plates with DMEM+10% FBS overnight, after cells reaching confocal state the D-peptide was added to the cultures at concentrations of 250 nM, 500 nM, 2000 nM, 3000 nM, 4000 nM or 8000 nM, and compared to a control treatment using vehicle solution (‘not treated’). Following 24-hour exposure to the D-peptide, cells where mixed with MTS solution (lOOul cells + 20ul working solution) in 96 wells plate and then incubated at 37°C for 2 hours, before absorbance was measured at 490nm wavelength.

[0271] Results - As can be seen in FIG. 2, the isolated D-peptide (‘D-peptide’) inhibited viability or proliferation of LX2 cells in a dose-dependent manner as evident by the approximately 20%-25% reduction of the absorbance measured in wells treated with 250 nM - 8000 nM isolated D-peptide, compared to control wells (‘not treated’).

[0272] To conclude, D-peptide is capable of inhibiting human hepatic stellate cells proliferation / viability, in comparison to untreated cells.

[0273] Example 3: D-peptide Stability in Human Serum ex vivo

[0274] The stability of the D-peptide was assessed in human serum, in comparison to the stability of the L-peptide.Methods - Briefly, human serum was obtained from a healthy donor, i.e., a donor with no indication or symptoms of liver disease or cancer, and immediately incubated at 37°C. New vials of the D-peptide and the reference L-peptide were dissolved in LCMS / MS Grade Methanol to a concentration of 20,000 nM. Thereafter, each peptide was mixed with human serum at a 2:1 ratio and incubated at 37°C as indicated - i.e., for 0, 5, 10, 30, 60, or 120 minutes (FIG. 3A); 0, 0.5, 1, 2, 4, 6, or 24 hours (FIG. 3B), or 0, 2, 3, 6, 12, or 14 days (FIG. 3C). After incubation, the peptide mixtures were centrifuged at 13,000 RPM for 10 minutes, and the supernatant obtained for LCMS / MS (Liquid chromatography-mass spectrometry) analysis. Standards of 10,000, 5000, 1000, 500, 100, and 10 nM were prepared with the same peptides to obtain a calibration curve.

[0275] Results - As detailed below, FIGs. 3A-3C are indicative of the isolated D-peptide of SEQ ID NO: 2 being characterized by improved serum stability compared to the reference L-peptide of SEQ ID NO: 3, as well as by robust ability to tolerate the harsh conditions of human serum including degrading enzymes, showed by keeping significant concentrations even after two weeks incubation in such conditions.

[0276] Advantageously, as can be seen in FIG. 3A, the concentration of the D-peptide remained steady during at least 120 min of incubation at 37°C in human serum, while the concentration of the L-peptide dropped very rapidly and sharply. As shown, by the first 10 min a decrease of more than 2-fold was detected (i.e., a drop from an initial concentration of approximately 5000 nM to approximately 1500 nM at 10 min). Moreover, after 30-40 min the concentration of the L-peptide continued to drop, while the concentrations of the D-peptide remained high and steady (approx. 5000 nM) for at least 120 min.

[0277] In addition, as can be seen in FIG. 3B, this trend continues for at least 24 hours of incubation in human serum, wherein the D-peptide maintains its initial concentrations. In fact, the figure is indicative of a difference of more than 20-fold in the concentration of the two peptides after 2 hours, a difference that maintains itself for a period of at least 24 hours at 37°C in human serum.

[0278] In FIG. 3C, the stability of the D-peptide is also evident from its surprising ability to keep an essentially constant peptide concentration, shown by a less than about20% decrease, during incubation in human serum for a period of up to 2-6 days at a temperature of about 37°C, compared to an initial concentration of the D-peptide.

[0279] Surprisingly, the concentration (nM) of the isolated D-peptide decreased by less than two-fold (less than 50%) after incubation of up to 6-14 days in human serum at a temperature of about 37°C, compared to the initial concentration of the D-peptide.

[0280] Considering the above, the half-life of the D-peptide is expected to be at least about 1-14 days, in human serum.

[0281] To conclude, the D-peptide exhibits superior stability in human serum compared to the reference L-peptide, while retaining substantially steady concentrations of less than 20% decrease in its initial concentrations up to day 6 in human serum and less than 50% decrease up to day 14 in human serum, suggesting it has a remarkable ability to withstand enzymatic degradation under these conditions.

[0282] Example 4: D-peptide half-life in vivo

[0283] The isolated D-peptide half-life was estimated in vivo in healthy mice and in model mice for liver fibrosis, namely, carbon tetrachloride (CC14)-induced acute hepatic fibrosis model mice.

[0284] Briefly, healthy mice were administered IV, IM or SC, with 5mg / Kg D-peptide and the concentration of the D-peptide was determined in blood of mice at varying time points.

[0285] For model mice- C57BL / 6J mice at 8 weeks of age were treated with CC14 and with the isolated D-peptide of SEQ ID NO: 2 injected in parallel. The concentration of the peptide injected to CC14-treated mice was 5mg / Kg. Blood was drawn from the mice tail at different time points (i.e., at 0, 1, 2, 24 and 48 hours). For LCMS / MS analysis samples were prepared by taking one part serum and two parts LCMS / MS Grade Methanol, mixing and centrifuging at 13,000 RPM for 10 minutes, and obtaining the supernatant for analysis. A standard was prepared with the D-peptide to obtain a calibration curve, with standard points of 10,000, 5000, 1000, 500, 100, and 10 nM.

[0286] As shown in Fig.4A, the D-peptide exhibited a half-life of 1.36, 0.7 or 0.6 hours when administered IV, IM or S.C., respectively, in healthy mice. This translates toabout 45-90 minutes, indicating that the peptides exhibit an extended half-life, compared to standard / reference peptides having a half-life of 2-4 minutes.

[0287] As can be seen in FIG. 4B, the isolated D-peptide has a half-life of approximately 1.5 hours. Immediately, following the intravenous injection of 5mg peptide per kg mice, the peptide concentration in serum obtained from the CC14-treated mice at time zero was 400 nM. Surprisingly, after 24 hours the peptide could be detected at concentrations of 83.6 nM and after 48 hours at concentrations of 4.7 nM. Even after 72 hours the peptide could still be detected at concentrations of 0.3 nM (data not shown).

[0288] Example 5 -D-peptides stimulate the activity of NK cells and reduce the activity of LX2 cells, in monocultures.

[0289] D-peptides of SEQ ID NOs: 1-2 and 4-5 were screened for their potential inhibitory effect on NLGN4-Nrxip PPI by measuring the activity of human natural killer (NK) cells and / or of human hepatic stellate cells (LX2), in monocultures, in comparison to the effect of the reference L-peptide of SEQ ID NO: 3.

[0290] D-peptides are assayed for their effect on the activity of NK cells by measuring the levels of CD 107a activation marker, and the effect on LX2 cells is evaluated by measuring aSMA levels. Monocultures were stimulated with increasing concentrations of the D-peptides.

[0291] The results are presented in Fig. 5, showing the effect of all-D peptide (i.e., having SEQ ID NO: 2), on monocultures of NK cells. The bar chart shown in Fig. 5, compares NK degranulation (CD107a%) in mono-culture between untreated NK cells (NK + NT) and the d-peptide-treated NK group (at 4000 ng / mL).

[0292] The untreated NK group shows -40% CD 107a expression, representing baseline activity. The d-peptide treated group shows a marked increase to about 75% CD 107a, indicating significantly enhanced NK activation. The difference between the two groups is substantial and statistically significant (***), demonstrating that the d-peptides greatly augment NK cell degranulation.Example 6 -D-peptides synergistically deactivate co-cultured LX2 cells through activated NK cells.

[0293] To test if NLGN4-Nrxip PPI interfering D-peptides mediated deactivation of LX2 cells is more prominent in the presence of NK cells, a co-culture setting is established for LX2 cells in the presence or absence of activated NK cells. Expression of aSMA is determined.

[0294] Example 7 -D-peptides modulate NLGN4X / B-neurexin axis by functionally reducing hepatic stellate cell activation and increasing NK cells adherence-mediated killing of hepatic stellate cells

[0295] To further test the effect of NLGN4-Nrxip PPI interfering D-peptides on the NLGN4X / p-neurexin axis different aspects of the LX2 / NK intercellular interaction are investigated. The tendency of NLGN4-Nrxip PPI interfering D-peptide-activated NK cells to physically interact with different populations of LX2 cells is assayed by FACS-sorting, testing only the adherent portion of cells in the co-culture of LX2 / peptide-activated-NK cells. The effects of increasing concentrations of D-peptides on two populations of cells are assessed based on sorting of the NK cell-specific CD56+ marker and the hepatic stellate cell activation marker aSMA, as either double-positive CD56+ / aSMA+ or single positive CD56+ / aSMA-.

[0296] Example 8 - Effects of D-peptides in-vivo, in liver-damage induced animal models Male 10-12-wk-old C57BL / 6J mice were treated with CC14 (0.5 pL / g body weight; 1:10 dilution with com oil) by IP injection twice weekly (morning dosing at 09:00). Injections were repeated for a total of 13 times (6 weeks). After 4 weeks of CC14 administration, these mice were divided into various treatment groups and were dosed by oral / IP / SQ injection. Body weight and feed intake was monitored twice a week.

[0297] At the end of treatment (Day-42), animals were sacrificed, plasma samples and organs weights were recorded at termination and liver tissue samples were collected for the estimations.

[0298] -Liver fibrosis markers: Protein levels (Western blot): a-SMA, -Neurexin and Lysosomal-associated membrane protein-1 (LAMP-1);-Liver fibrosis markers by mRNA (RT-PCR): aSMA, -Neurexin, TNFa, IL-6, MCP-1, TIMP-1, Collagen la;

[0299] -NK activation markers (Blood) (Flow cytometry analysis): CD107a;

[0300] -Remining portion of liver was used to carry out Histopathology analysis to determine the effect of treatment on fibrosis, necrosis and inflammatory infiltration by following staining:

[0301] -H&E - Hematoxylin and Eosin (used for general tissue structure and morphology) -Sirius Red - Stains collagen fibers, often used to assess fibrosis.

[0302] -Quantification of liver fibrosis, inflammation and injury is performed using following scales:

[0303] Sirius Red: 0-4 scale: 0, no fibrosis; 1, minimal portal fibrosis; 2, portal fibrosis with septa formation.; 3, localized bridging fibrosis; 4, extensive bridging fibrosis.

[0304] H&E staining: 0-3 scale for inflammation (H & E stain): 0, no inflammation; 1, mild inflammation; 2, moderate inflammation; and 3, severe inflammation. A 0-3 scale for degeneration (based on intralobular degeneration) (H & E stain): 0, no degeneration; 1, mild degeneration (acidophilic bodies, ballooning degeneration and / or scattered foci of hepatocellular necrosis in 1 / 3 of lobules or nodules); 2, moderate degeneration (involvement of 1 / 3-2 / 3 of lobules or nodules); 3, severe degeneration (involvement of >2 / 3 of lobules or nodules). For other findings: 0, No Abnormality Detected; 1, Minimal; 2, Mild; 3, Moderate; 4, Severe.

[0305] Remaining parts of the liver are flash frozen in liquid nitrogen and preserved for further analysis.

[0306] Results:

[0307] Effect on body weight: The results show that the treatments did not have an effect on body weight: CC14 control animals (26 ± 0.1 g) did not show significant difference in body weight as compared to naive control animals (26.3 ± 0.3 g) from day 1 to day 28 (induction phase). Untreated animals (27 ± 0.3 g), or animals treated with Resmetirom, varying doses of D-peptide, administered by either i.p. or s.c. routes didnot show significant changes in body weight as compared with negative control, from day 28 to day 42.

[0308] Effect on cumulative food consumption: CCh control animals (28.8 ± 0.5 g) showed significant reduction in cumulative food consumption as compared to naive control animals (33.8 ± 0.5 g) from day 1 to day 26. Untreated animals (17.3 ± 0.6 g), or animals in the treatment groups did not show significant changes in cumulative food consumption as compared with negative control from day 29 to day 40.

[0309] Effect of treatment: in order to determine the effect of the D-peptide on inflammatory markers and anti-inflammatory activity of the d-peptide, the levels of AST (Aspartate Aminotransferase) and ALT (Alanine Aminotransferase) enzymes were determined under the various experimental settings. Generally, AST is not specific to the liver, whereas ALT is secreted from the liver into the blood when the liver is inflamed, but high levels of both enzymes correlate with liver damage (such as inflammation and fibrosis).

[0310] To this aim, the CCL4 experimental model included 4 weeks of fatty liver induction (by CCL4) followed by 2 weeks of treatment, of i.p. or s.c. administration of D-peptide. This short treatment duration can demonstrate if the tested D-peptide can reduce, treat or reverse liver damage, for example, by reducing or even halting the inflammation process.

[0311] Measuring AST blood levels in treated mice demonstrated that, advantageously, the levels of AST did not increase during the short treatment with the D-peptide (not shown).

[0312] Further, as demonstrated in Fig. 6, ALT levels were advantageously and surprisingly reduced, when the animals were treated with the D-peptide (Luji-4), indicating the enhanced effect of the D-peptide in treating or even preventing (in-view of the short treatment time, after CCL4 damage induction) inflammation and thus liver damage. As shown in Fig. 6, D-peptide treatment groups clearly exhibited a reduction in ALT levels as compared with negative control.

[0313] Collectively, these results clearly demonstrate the anti-inflammatory, in-vivo effect of the D-peptide.The effect of treatment on organ weight: for both liver and spleen, the various treatment groups did not exhibit significant changes in liver or spleen weight as compared with negative control.

[0314] Effect of D-peptide treatment on various liver mRNA biomarkers is shown in Figs. 7A-7C. MCP-1 - D-peptide administered s.c., exhibited significant reduction in MCP-1 expression as compared with negative control, while negative control showed significant increase in MCP-1 expression as compared with untreated animals. (Fig.

[0315] 7A); TIMP-1 - D-peptide exhibited reduction in TIMP-1 expression as compared with negative control, whereas negative control showed significant increase in TIMP-1 expression as compared with untreated animals (Fig. 7B); IL-6 - D-peptide treatment exhibited reduction in TIMP-1 expression as compared with negative control (Fig. 7C).

[0316] The effect of treatment on liver protein biomarkers is shown in Figs. 8A-8C (showing quantification of Western Blot analysis): P-Neurexin- Treatment groups did not show significant changes in P-Neurexin expression as compared with negative control, whereas negative control showed significant increase in P-Neurexin expression as compared with untreated animals (Fig. 8A); aSMA - Treatment groups exhibited changes in aSMA expression as compared with negative control, whereas negative control showed trend increase in aSMA expression as compared with untreated animals (Fig. 8B); LAMP-1 - Treatment groups exhibited changes in LAMP-1 expression as compared with negative control, whereas negative control showed trend increase in LAMP-1 expression as compared with untreated animals (Fig. 8C).

[0317] Next, the effect of D-peptide treatment on liver histopathology was determined. As shown in Figs. 9A-9D, D-peptide treatment groups exhibited changes in fibrosis score (Fig. 9A), inflammation (Fig. 9B), and hepatocyte necrosis (Fig. 9C), compared with negative control. Negative control groups exhibited significant increase in fibrosis score, inflammation (Fig. 9B) and hepatocyte necrosis (Fig. 9C), as compared to untreated animals.

[0318] With respect of hepatocyte vacuolation / degeneration rarefaction (Fig. 9D) - D-peptide treatment groups showed significant reduction in hepatocyte vacuolation / degeneration rarefaction score as compared with negative control. Negative control showed significant increase in hepatocyte vacuolation / degeneration rarefaction score as compared with untreated animals.Likewise, with respect of hepatocyte cytoplasmic rarefaction, negative control showed significant increase in hepatocyte cytoplasmic rarefaction score as compared with untreated animals. Some of the D-peptide treatment groups showed reduction in hepatocyte cytoplasmic rarefaction score as compared with negative control.

[0319] Next, the effect of the treatments on NK cells, as determined by flow cytometry blood analysis as determined. As shown in Fig. 10, D-peptide treatments showed significant increase in CD 107a expression as compared with negative control.

[0320] Collectively, the results demonstrate the in-vivo effect of the administered D-peptides on liver fibrosis.

[0321] The carbon tetrachloride (CC14)-induced liver fibrosis mouse model was used as an experimental system for investigating hepatic fibrogenesis. Repeated administration of CCh causes sustained hepatocellular injury, oxidative stress, and inflammatory responses, which activate hepatic stellate cells and drive excessive extracellular matrix deposition. These pathological changes closely resemble the progression of human liver fibrosis, making the model highly relevant for mechanistic studies and preclinical evaluation of anti-fibrotic agents. As demonstrated herein, histological evidence, such as collagen accumulation detected by Sirius Red staining and increased a-SMA expression, along with elevated serum transaminases, confirms the severity of fibrosis.

[0322] As demonstrated herein, animals treated with CC14 showed significant increase in AST and ALT as compared with untreated animals, being indicative of development of liver fibrosis. Treatment with D-peptide (e.g., LUJI-4 ,5 & 30 mg / kg, TIW, IP; and LUJI-4 5, 15 & 30 mg / kg, TIW, SC) showed a trend towards reduction in AST as compared with negative control. LUJI-4 (5 & 30 mg / kg, TIW, IP) and LUJI-4 (30 mg / kg, TIW, SC) showed significant reduction in ALT as compared with negative control. LUJI-4 (5 & 15 mg / kg, TIW, SC) showed trend towards reduction in ALT as compared with negative control. This indicates a beneficial effect of LUJI-4 in reducing elevated liver enzyme levels. This is further supported with LUJI-4 (15 mg / kg, TIW, SC) showing significant reduction and LUJI-4 (5 & 30 mg / kg, TIW, IP) and LUJI-4 (5 & 30 mg / kg, TIW, SC) showing trends toward reduction in Inflammation as compared with negative control animals. LUJI-4 (5 & 30 mg / kg, TIW, IP) and LUJI-4 (5, 15 & 30 mg / kg, TIW, SC) showed trend towards reduction in hepatocyte necrosis ascompared with negative control animals. LUJI-4 (5 & 30 mg / kg, TIW, IP) and LUJI-4 (5, 15 & 30 mg / kg, TIW, SC) also showed trend towards reduction in Hepatocyte cytoplasmic rarefaction as compared with negative control animals. LUJI-4 (15 mg / kg, TIW, SC) showed significant reduction in hepatocyte vacuolation / degeneration Rarefaction.

[0323] Evaluation of the liver tissues for mRNA levels as well as protein levels demonstrated that, for example, LUJI-4 (5 & 30 mg / kg, TIW, IP) showed a trend towards reduction in aSMA and P-Neurexin mRNA levels in liver tissue as compared to negative control animals. LUJI-4 (5, 15 & 30 mg / kg, TIW, SC) showed a dose-related trend towards reduction in aSMA and P-Neurexin mRNA levels in liver tissue as compared to negative control animals. LUJI-4 (5 & 30 mg / kg, TIW, IP) and LUJI-4 (5, 15 & 30 mg / kg, TIW, SC) showed a trend towards reduction in mRNA levels of liver IL-6 levels in a dose-related manner indicating improvement in inflammation, further, LUJI-4 (5 & 30 mg / kg, TIW, IP) and LUJI-4 (5, 15 & 30 mg / kg, TIW, SC) showed a dose-related trend towards reduction in aSMA, P-Neurexin and LAMP-1 protein levels as seen from Western blot analysis. Additionally, LUJI-4 at 30 mg / kg, TIW, IP, LUJI-4 at 5 mg / kg, TIW, SC and LUJI-4 at 15 mg / kg, TIW, SC showed significant increase in CD107a cells as compared to negative control animals.

[0324] Overall, treatment with the D-peptide (e.g., LUJI4) has halted further deterioration of liver fibrosis by showing reduction in inflammatory marker like IL-6 and improvements in liver histopathology. This is supported by similar results obtained from western blot analysis. Accordingly, the D-peptide treatment has a substantive effect on reduction of liver fibrosis.

[0325] Example 9 - D-peptide in vivo effect on D-neurexin expression of HSCs and F-actin expression of liver NK cells

[0326] P-neurexin expression of HSCs and F-actin expression of liver NK cells are evaluated in mice induced for acute liver fibrosis to indicate if the NLGN4-Nrxip PPI interfering D-peptides deactivates HSCs through inhibition of P-neurexin And restoration of NK activity through increased expression of F-actin, which is necessary for normal cellular function and motility.Example 10 - D-peptide effect on proliferation of Hepatocellular carcinoma (HCC) cells in vitro

[0327] The effect NLGN4-Nrxip PPI axis modulation exerts on proliferation and carcinogenicity of liver cancer cells is evaluated in-vitro in the Hepatocellular carcinoma (HCC) cell line Hep3B, by exposing the cells to 8000ng / ml of D-peptides and performing gene expressions analyses of P-neurexin and of specific proliferation markers and analysis of the stage of cell cycle.

[0328] Expression of a-feto-protein (a-FP), a marker for carcinogenicity, and of Carboxy Fluoroscein Succinimidyl Ester (CSFE), PDGFRA and MKI67 markers for proliferation, are evaluated in Hep3B cells treated with the D-peptides or the reference L-peptide. ELISA and flow cytometry analyses are performed to indicate any reduction in the expression levels of all markers following treatment with D-peptides.

[0329] To ratify attenuation of the proliferative capabilities of HCC cells following modulation by D-peptides, the stage of the cell cycle of Hep3B cells treated with D-peptide 1 or with a reference L-peptide is evaluate using flow cytometry and Propidium Iodide (PI) -staining.

[0330] Example 11 -D-peptide effect on cellular proliferation and / or oncogenic signaling of Hepatocellular carcinoma (HCC) cells in vitro

[0331] To further substantiate the ability of the D-peptides to attenuate proliferative and / or oncogenic properties of Hep3B HCC cells, the level of activated markers belonging to the Akt- and mTOR- signaling pathways downstream of P-neurexin is evaluated. Level of expression of phosphorylated AKT / mTOR / P70S6K is evaluated using flow cytometry in Hep3B cells treated with D-peptides or with the reference L-peptide.

[0332] Example 12 - D-peptide effect on cell death of Hepatocellular carcinoma (HCC) cells in vitro

[0333] To assess whether the ability of the D-peptides to attenuate cell division and activate oncogenic signaling pathways is accompanied with a change in cell death and viability, the effect of inhibiting NLGN4-Nrxip PPI using D-peptides is evaluatedusing markers for viability and cell death, including markers indicative of apoptosis and necrosis such as Annexin-V+ / PI and CDNK2A, respectively.

[0334] Example 13 -Effect of D-peptide on various types of cancer cells

[0335] The effect of the D-peptides on various cancer cells, including, effect on cell division, cell death and / or cell viability is evaluated using various markers and / or assays indicative of apoptosis and / or necrosis.

[0336] The tested cells include:

[0337] - Colorectal cancer cells, including Caco2 (cell model, a clone of colorectal adenocarcinoma cells from human);

[0338] - Breast Cancer cells, including, MCF-7 cells (human breast cancer cell line with estrogen, progesterone, and glucocorticoid receptors);

[0339] - Prostate cancer cells: PC3 cells (a human androgen-independent prostate cancer cell); - Cholangiocarcinoma cells;

[0340] - Liver metastasis cells.

[0341] Example 14 - Single dose pharmacokinetic study of D-peptides following three different route of administration in male C57BL / 6 mice

[0342] The objective of this study is to determine the plasma pharmacokinetics of D-peptides, specifically, the isolated D-peptide having an amino acid sequence as denoted by SEQ ID NO: 2 [H-( dM )( dE )( dQ )G( dE )( dF )( dL )( dN )( dY )( dD )-OH], in three different routes of administration in male C57BL / 6 mice.

[0343] 1. Materials and Methods:

[0344] Test item: D-peptide (for example of SEQ ID NO: 2) with a molecular weight of: 1108.34 (free base) and / or 1245.33 (salt).

[0345] Test system: 27 male C57BL / 6 mice, 9 mice / group.

[0346] Dose Formulation Preparation:

[0347] The dose formulation is prepared freshly on the day of dosing.

[0348] Formulation vehicle: DMSO: PBS (5% DMSO IN PBS)Procedure for Dose Formulation Preparation (Solution):

[0349] For preparation of a lOmL D-peptide solution at a net concentration of Img / mL weigh 11.2 mg of peptide into a 15-20 mL transparent type I glass vial / bottle. Add 0.5g DMSO to the vial and mix gently, than immediately add 9.5g of PBS and mix gently. Vortex for approximately 30 seconds and confirm complete peptide dissolution by visually inspecting the vial. In case a clear transparent solution is not observed, repeat additional 30 seconds vortex cycles until complete dissolution. Cover the vial with an aluminum foil.

[0350] Final formulation concentration is made as mentioned in the Group allocation and treatment table 1 below.

[0351] Table 1 - Group Allocation and Treatment:

[0352]

[0353] Dose administration:

[0354] Intravenous (IV)

[0355] Intramuscular (IM)

[0356] Subcutaneous (SC)

[0357] 2. Blood collection and storage of samples:

[0358] Site of blood collection: Retro orbital sinus puncture

[0359] Anticoagulant: K2EDTA (20 pL of 200 mM solution per 1 mL of blood)

[0360] Blood collection time points: 0, 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24h post dose (Total 9 time-points)

[0361]

[0362]

[0363] Similar patern is followed for groups 2 and 3.

[0364] Volume of blood collected: ~0.2 mL at each time points from each mouse. Blood sampling procedure: Sparse sampling

[0365] Blood centrifugation time: 4000 rpm for 10 minutes at 4 °C

[0366] Plasma storage: -70 ± 10 °C until analysis

[0367] Plasma labeling details: Study number, Group, Animal ID and Time point.

[0368] Note:

[0369] 1. Blood samples are kept on wet ice until centrifugation. Centrifugation is done within 30 minutes of collection and plasma is checked for hemolysis.

[0370] 2. Prepared dose formulation is discarded after the completion of in-life phase.

[0371] 3. On completion of last blood sampling, animals are sacrificed, and carcasses discarded.

[0372] 3. Bioanalysis:

[0373] Matrix: Plasma

[0374] Analyte: D-peptide of SEQ ID NO: 2

[0375] Bioanalysis is performed by frt-for-purpose analytical method using LC- MS / MS.

[0376] Upon finalization of study report, remaining biological study samples are discarded.

[0377] 4. pharmacokinetic data analysis and evaluation:Pharmacokinetic Software Phoenix® Software, version 8.3, USA.

[0378] Pharmacokinetic Parameters : Co, Cmax, Tmax, AUCo-t, AUCo-00, Kei, TI / 2, Vd,

[0379] Cl, Vd / F, Cl / F, MRTiast and

[0380] % bioavailability

[0381] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

CLAIMS:What is claimed is:

1. An isolated D-peptide comprising an amino acid (AA) sequence as denoted by SEQ ID NO: 1, wherein SEQ ID NO: 1 comprises at least one D enantiomeric AA (dAA).

2. The isolated D-peptide according to claim 1, consisting of an AA sequence as denoted by SEQ ID NO: 1, wherein SEQ ID NO: 1 comprises at least one dAA.

3. The isolated D-peptide according to claim 1 or 2, wherein SEQ ID NO: 1 comprises 2 to 9 dAA.

4. The isolated D-peptide according to any one of claims 1-3, wherein all 9 chiral amino acids (AAs) of SEQ ID NO: 1 are D-amino acid (all-dAAs), as denoted by SEQ ID NO: 2.

5. The isolated D-peptide according to any one of claims 1-4, wherein the isolated D-peptide is capable of reducing the activation of Hepatic Stellate Cells (HSCs).

6. The isolated D-peptide according to any one of claims 1-5, wherein the isolated D-peptide is capable of attenuating HSCs proliferation and / or viability.

7. The isolated D-peptide according to any one of claims 1-6, wherein the isolated D-peptide has an improved stability in human serum in comparison to a reference peptide that has an amino acid sequence as denoted by SEQ ID NO: 3; wherein all chiral amino acids (AAs) of the reference peptide are L-amino acids (all-lAAs).

8. The isolated D-peptide according to any one of claims 1-7, wherein the isolated D-peptide is stable for at least 60 minutes at 37°C in serum.

9. The isolated D-peptide according to any one of claims 1-8, having a half-life of at least 3 days in serum.

10. The isolated D-peptide according to any one of claims 1-9, having a half-life of at least about 30 minutes in vivo.

11. The isolated D-peptide according to any one of claims 1-10, wherein the isolated D-peptide is capable of affecting neuroligin 4 (NLGN4) - Neurexin i (Nrxip) protein-protein interaction.

12. A composition comprising an isolated D-peptide according to any one of claims 1-11, and at least one pharmaceutically acceptable carrier / s, excipient / s, auxiliaries, and / or diluent / s.

13. The isolated D-peptide according to any one of claims 1-11, or the composition according to claim 12, for use in treating, attenuating, and / or preventing progression of a liver disorder in a subject in need thereof.

14. The isolated D-peptide according to claim 13, wherein the liver disorder is selected from: fibrosis, Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) , Metabolic Dysfunction-Associated Steatohepatitis (MASH), cirrhosis, hepatitis, viral hepatitis, liver adenoma, insulin hypersensitivity, liver cancer, liver metastasis, or any combination thereof.

15. The isolated D-peptide according to claim 14, wherein the liver disorder is liver fibrosis or liver cancer.

16. The isolated D-peptide according to any one of claims 1-11, or the composition according to claim 12, for use in treating, attenuating, and / or preventing progression of a cancer condition in a subject in need thereof.

17. The isolated D-peptide according to claim 16, wherein the cancer is selected from liver cancer, liver metastasis, cholangiocarcinoma, colorectal cancer, breast cancer and prostate cancer.

18. The isolated D-peptide or according to any one of claims 13-17, wherein the isolated D-peptide or the composition are formulated for systemic administration.

19. The isolated D-peptide according to any one of claims 13-18, wherein the isolated D-peptide or the composition for use is administered in combination with at least one additional therapeutic agent.

20. The isolated D-peptide according to any one of claims 1-11, or the composition according to claim 12, for use in reducing activation of HSCs.

21. A natural killer (NK) cell comprising the isolated D-peptide according to any one of claims 1-11, or the composition according to claim 12.

22. The NK cell according to claim 21, wherein the NK cell is capable of reducing or inhibiting activity of HSCs.

23. A composition comprising the NK cell according to claims 21 or 22.

24. The NK cell according to any one of claims 21-22, or the composition according to claim 23, for use in treating, attenuating and / or preventing progression of a liver disorder in a subject in need thereof.

25. The NK cell or according to claim 24, wherein the liver disorder is selected from: fibrosis, Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD), Metabolic Dysfunction-Associated Steatohepatitis (MASH), cirrhosis, hepatitis, viral hepatitis, liver adenoma, insulin hypersensitivity, liver cancer, liver metastasis, or any combination thereof.

26. The NK cell according to claim 25, wherein the liver disorder is liver fibrosis or liver cancer.

27. The NK cell according to any one of claims 21-22, or the composition according to claim 23, for use in treating, attenuating and / or preventing progression of cancer in a subject in need thereof.

28. The NK cell according to claim 27, wherein the cancer is selected from: liver cancer, liver metastasis, cholangiocarcinoma, colorectal cancer, breast cancer and prostate cancer.

29. The NK cell e according to any one of claims 24-28, wherein the cell or the composition comprising the same is administered systemically.

30. The NK cell according to any one of claims 24-29, wherein the cell or the composition comprising the same is administered in combination with at least one additional therapeutic agent.

31. A method of treating, attenuating and / or preventing progression of a liver disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of the isolated D-peptide according to any one of claims 1-11, or the composition according to claim 12.

32. A method of treating, attenuating and / or preventing progression of a liver disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of the NK cell according to any one of claims 21-22, or the composition according to claim 23.

Images